Mechanistic Investigation of Cryptosporidium Oocyst Attachment to Solid Surfaces

Abstract Cryptosporidium parvum (C. parvum) causes potentially life-treating gastrointestinal disease in humans and is not treated by conventional water filtration and disinfection methods. The current available techniques for oocyst detection and quantification are not economical, thus limiting the ability of water utilities to understand the fate and transport of C. parvum oocysts through watershed. Moreover, natural surfaces, such as biofilms, can bind pathogens such as C. parvum oocysts, further complicating the fate and transport question, as the exact oocyst attachment mechanisms and binding forces to the substrates are not fully understood. For this reason, we studied adhesion using biophysical and analytical tools, and developed a device with Cell-Impinted Polymer (CIP) surface to specifically and selectively adhere oocysts. For the first step, we investigated the affinity of the C. parvum oocyst to a series of model functional groups (-OH, -NH2 and -COOH) using atomic force microscopy (AFM) with single-cell force spectroscopy technique. Binding forces were measured over several weeks, to consider the influence of age on biophysical interactions in various water chemistry conditions. This work demonstrated calcium-mediated mechanism for oocysts binding that underwent changes during aging of the oocysts. In addition to water chemistry and surface functional groups, the effect of aging of the oocysts on their mechanical properties was studied by applying Johnson-Kendall-Roberts (JKR) contact mechanics model to the AFM retraction curves to account for the Young's modulus of oocysts wall. A significant portion of the obtained force curves were fitted with the applied JKR model for several weeks. Thus, we hypothesis that the effective Young’s modulus of oocyst wall is reduced with respect to aging which represents the loss of the communicative coupling of lipid bilayer or protein denaturation in inner layer of the wall. For the next step, the information obtained from this work may be used to guide the engineering of biomimetic surfaces to capture the oocyst in the environment. As it mentioned earlier, a major challenge for C. parvum monitoring in water systems is the expensive, time consuming techniques currently used in EPA method. The proposed CIP surface described in this work is a preliminary exploration of a potential strategy that could streamline the process by providing cost effective, shelf stable capture devices that can be used for in situ immunofluorescence detection of C. parvum oocysts. For future, the CIP surface can be further investigated for detection of the oocysts quantitatively to predict C. parvum oocyst fate and transport in the watershed. Converting the designed surfaces into point-of-use or rapid laboratory devices would need the development of microfluidics chips in combination with immunofluorescence assay. In addition, the potential use of chemically fixed oocysts as a template will reduce the biological risk of providing live oocysts, expense and also batch to batch variation in oocysts characteristics. The impact of factors on reproducibility, selectivity as well as stability, can also be further investigated in the environmental conditions to confirm oocyst specific detection by CIP surface. Additionally, the aging mechanism of oocyst’s wall should be considered in future work.