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Description
Mucus is comprised of mucin glycoproteins that protects the underlying epithelia against infection. This barrier is colonized by a plethora of microbes and viruses that infect bacteria, known as bacteriophage. There is a general consensus that there are no intrinsic interactions between phage and eukaryotic cells. However, phages are known to be transported into the bloodstream and urine upon different types of administration, yet the mechanism(s) remain unknown. In vitro studies using T4 coliphage demonstrate that bacteriophage adherence to mucus (BAM) is mediated by immunoglobulin-like (Ig-like) domains via the immunogenic outer capsid (hoc) proteins that are involved in recognition and adhesion processes. Ig-like domains helps phage attach on mucin molecules. The first aim was to develop a phage purification method to remove bacterial lipopolysaccharides (LPS) from phage preparations in order to detect a cellular response to phage and not LPS. The second aim of this study was to probe the stimulation of a cellular response to phage using proteomics. Here, I developed the Phage On Tap method as a rapid, efficient and cheap way to create high titer homogeneous T4 phage banks. This process was compared with traditional existing techniques. The LPS data generated from this study showed no reduction of LPS levels using the traditional method, but the Phage On Tap method reduced it slightly. Although our method was unsuccessful in reducing the LPS, it was efficient for making high titer homogeneous phage stocks that were stored as a phage bank for common laboratory use that has led to more consistent results. Proteomic analyses revealed heat shock protein (HSP) 70 and 90 were abundant upon phage addition. HSP70 has been proposed to transport the MUC1 cytoplasmic tail (MUC1CT) to the mitochondria inhibiting apoptosis and HSP90 has been postulated to be cleaved and localize to the nucleus in complexes with b-catenin which is involved in gene transcription. We hypothesize that phage binding to MUC1 may play a role in the activation of transcription factors for mucin synthesis and protect the cell from apoptosis. This work provides initial insight into how phages interact with the human mucosal immune system.
