March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Characterization of a Truncated and More Potent Zinc Metalloprotease, ZmpC, from Streptococcus pneumoniae that Abrogates the MUC16 Barrier in Ocular Surface and Tracheal-Bronchial Epithelial Cells
Author Affiliations & Notes
  • Balaraj B. Menon
    Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
  • Bharathi Govindarajan
    Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
  • Sandra Spurr-Michaud
    Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
  • Ilene K. Gipson
    Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  Balaraj B. Menon, None; Bharathi Govindarajan, None; Sandra Spurr-Michaud, None; Ilene K. Gipson, None
  • Footnotes
    Support  R01EY18850 to IKG
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1844. doi:https://doi.org/
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      Balaraj B. Menon, Bharathi Govindarajan, Sandra Spurr-Michaud, Ilene K. Gipson; Characterization of a Truncated and More Potent Zinc Metalloprotease, ZmpC, from Streptococcus pneumoniae that Abrogates the MUC16 Barrier in Ocular Surface and Tracheal-Bronchial Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1844. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Our laboratory recently discovered a mechanism by which an epidemic conjunctivitis-causing strain of the gram-positive pathogen Streptococcus pneumoniae (strain SP168) invades ocular surface epithelial cells. Specifically, strain SP168 secretes a zinc metalloprotease, ZmpC, that cleaves the ectodomain of the membrane mucin MUC16 - an important defense component in the apical glycocalyx of ocular surface and tracheal-bronchial epithelial cells. The objective of this study is to characterize the ZmpC from strain SP168 and compare it to its counterpart from S. pneumoniae strain TIGR4 that is less efficient at cleaving MUC16.

Methods: : Sequencing of the SP168 zmpC gene was done at the University of Maine DNA Sequencing Facility. zmpC from strains SP168 and TIGR4 were cloned and expressed in E. coli 10G cells using the Expresso Rhamnose System (Lucigen Corp.). Recombinant ZmpC (rZmpC) was purified using Ni-NTA affinity chromatography. Potencies of the two isozymes were evaluated by their abilities to induce MUC16 ectodomain shedding in cultured human corneal (HCLE) and tracheal-bronchial (TrBr) epithelial cells. Following ectodomain cleavage, the amount of residual MUC16 on the surfaces of HCLE and TrBr epithelial cells was estimated by performing biotinylation assays.

Results: : DNA sequencing results revealed that the zmpC gene from S. pneumoniae strain SP168 is smaller than that of strain TIGR4 or other known zmpC sequences by 723 bp. Recombinant versions of both isozymes were functional as assessed by their abilities to induce MUC16 ectodomain shedding in HCLE and TrBr epithelial cells. However, rZmpC corresponding to strain SP168 was found to induce nearly twice as much MUC16 ectodomain shedding in comparison to that induced by rZmpC from strain TIGR4. Biotinylation results indicated that the surface abundance of MUC16 was decreased by close to 90% following treatment of HCLE and TrBr epithelial cells with SP168 rZmpC.

Conclusions: : Our results suggest that the epidemic conjunctivitis-causing S. pneumoniae strain SP168 harbors a truncated and more potent form of ZmpC, which abrogates the MUC16 barrier at the ocular surface. This feature may explain the increased virulence of strain SP168 and its ability to trigger a more aggressive infection.

Keywords: microbial pathogenesis: experimental studies • cornea: epithelium • cornea: surface mucins 
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