At the surface of the eye, infections occur via breaks in the surface mucin barrier or through the mucin barrier. Examples of the first are Staphylococcus aureus (SA) or Streptococcus pneumoniae (SPE) (encapsulated) keratitis that occurs as the result of epithelial surface damage from trauma. Other types of infection, e.g., outbreaks of bacterial conjunctivitis from non-encapsulated Streptococcus pneumoniae (SPN), occur without evidence of ocular surface damage. Most studies of host-pathogen interactions have used models that involve the physical induction of epithelial damage prior to infection, allowing a pathogen to cross the apical mucosal mucin barrier. In contrast, there is limited understanding of how the mucin barrier can be manipulated or compromised directly by epidemic-causing pathogens to facilitate infection. We propose to compare how SA, SPE (opportunistic organisms), and nontypeable, non-encapsulated SPN (organisms capable of causing epidemic conjunctivitis in the absence of known or suspected ocular surface trauma), interact with and affect membrane-associated mucins to gain access to epithelial cells. Studies will use corneal and conjunctival epithelial cell lines expressing apically the glycosylated membrane-associated mucins found in native epithelia (MUC1, 4 and 16) and 3 clinical isolates-SA, SPE, and SPN-all derived from ocular surface infections.
Our Specific Aims are:
Aim I : Compare ectodomain release, cell surface density and glycosylation characteristics of MAMs MUC1, MUC4 and MUC16 on human corneal and conjunctival epithelial cells, in response to culture with the two opportunistic bacteria, SA and encapsulated SP, and the epidemic-causing non-encapsulated strain of SP, and their exoproducts.
Aim II : Determine the mechanism by which non-encapsulated SP exoproducts induce release of the membrane mucin MUC16.
Aim III : Determine the extent to which membrane mucins present an obstacle to bacteria or their exoproducts to reach the epithelial surface membrane by comparing (1) cytopathic effects and internalization rates for SA and the SP strains into control human corneal-limbal epithelial (HCLE) cells, and into HCLE cells in which MAM expression is abrogated by siRNA, or in which glycosylation of mucins is blocked, and (2) by comparing effects of SA, SPE, or SPN, or their exoproducts on expression of stress-induced cytokines IL-6, IL-8 and TNF-? by HCLE cells and in HCLE cells in which MAM expression is abrogated or in which glycosylation of the mucins is altered.
Aim I V: Determine if the three types of bacteria bind to specific released mucins, MUCs 1, 4 and 16, and/or secreted goblet cell mucin MUC5AC present in the tear film, and whether such binding acts as a protective mechanism, preventing adhesion to HCLE cells in control, membrane-associated mucin knockdown or deglycosylated mucin conditions
Infection remains a leading cause of morbidity and mortality worldwide, and most infections originate at a mucosal boundary-the mucosal surface of the respiratory tract, gastrointestinal tract, urogenital tract or the eye. Mucins on the wet-surfaced mucosa are hypothesized to provide a barrier against continuous exposure to trillions of microbes, and preliminary data from our laboratory indicate that, of the two types of mucins on these epithelia, secreted or membrane associated, it is the membrane-associated mucins integral to surface membranes that form this cellular barrier. The research proposed in this application will provide new information about how bacteria that cause epidemic conjunctivitis are more capable of crossing the membrane mucin barrier than are the opportunistic pathogens and, in doing so, will provide clues for treatment alternatives.
|Menon, Balaraj B; Zhou, Xiaohong; Spurr-Michaud, Sandra et al. (2016) Epidemic Keratoconjunctivitis-Causing Adenoviruses Induce MUC16 Ectodomain Release To Infect Ocular Surface Epithelial Cells. mSphere 1:|
|Menon, B B; Kaiser-Marko, C; Spurr-Michaud, S et al. (2015) Suppression of Toll-like receptor-mediated innate immune responses at the ocular surface by the membrane-associated mucins MUC1 and MUC16. Mucosal Immunol 8:1000-8|
|Gipson, Ilene K; Spurr-Michaud, Sandra; Tisdale, Ann et al. (2014) Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function. PLoS One 9:e100393|
|Menon, Balaraj B; Govindarajan, Bharathi (2013) Identification of an atypical zinc metalloproteinase, ZmpC, from an epidemic conjunctivitis-causing strain of Streptococcus pneumoniae. Microb Pathog 56:40-6|
|Govindarajan, Bharathi; Menon, Balaraj B; Spurr-Michaud, Sandra et al. (2012) A metalloproteinase secreted by Streptococcus pneumoniae removes membrane mucin MUC16 from the epithelial glycocalyx barrier. PLoS One 7:e32418|
|Govindarajan, Bharathi; Gipson, Ilene K (2010) Membrane-tethered mucins have multiple functions on the ocular surface. Exp Eye Res 90:655-63|