Microsporidia are an obligate, intracellular fungal pathogen that can cause devastating, disseminating infections in the immunocompromised. Because of the limitations of current medications, microsporidia’s abundant presence in the environment, and an increasing number of at-risk populations, investigation into decreasing microsporidia infectivity is needed. As an intracellular pathogen, microspridial attachment is a vital first step to infection, and if attachment is reduced, previous work shows that infectivity is mitigated. An in silico analysis of Encephalitozoon intestinalis revealed a predicted protein similar in sequence to ADAM (A Disintegrin And Metalloproteinase) proteins. This predicted protein is termed microsporidia ADAM or MADAM. ADAM proteins contain an integrin binding region, which is well known to bind to integrin proteins. Integrins are important receptors for attachment and cell signaling, and several pathogens utilize host integrins as a receptor to aid in attachment during infection. Immunoelectron microscopy demonstrates that MADAM protein is found on the plasma membrane, anchoring disk, and polar tube of E. intestinalis spores. Our hypothesis is that MADAM is involved in the key role of host cell attachment. To this end, a 17 amino acid long section of the MADAM protein was generated that surrounded the integrin binding domain. During spore adherence assays, pretreating host cells with this small peptide protein, significantly decreased E. intestinalis spore attachment to host cells as compared to control samples. These results suggest E. intestinalis cleverly exploits host integrins as a means to bind to host cells before infection.

Microsporidia are a distant fungal pathogen that have severe clinical consequences for the immunocompromised. Previous work identified a microsporidian pathogen protein termed Microsporidian ADAM or MADAM. This protein has close sequence homology to other ADAM proteins (A Disintegrin and Metalloproteinase) in two microsporidian species, Encephalitozoon intestinalis and E. cuniculi. ADAM proteins have a wide range of functions, including binding to integrins and host signaling. It is known that many pathogens manipulate integrins to invade host cells, and it is predicted that microsporidia are also exploiting this host target. Previous work with the MADAM protein demonstrated that this protein has a role in adherence to host cells. Separate work showed integrin inhibitors can also decrease spore adherence to cells. Experiments in this project complement previous research and further characterize the binding of microsporidia to host integrins and the intracellular consequences of that binding. This work found the integrin binding sequence of MADAM (MADAM peptide) is important for spore binding to host cells. Separate work shows that the host β1 integrin is also involved in spore adherence. Additional work demonstrated that spores and the MADAM peptide elicited an increase in host integrin signaling in Western blotting experiments. And finally, preliminary acellular interferometry experiments suggest the MADAM protein binds specifically to α5β1 and α6β4 integrins. Together, these results suggest microsporidia spores rely, in part, on host integrins to bind to host cells before infection.