Investigation of the molecular and mechanistic basis for attachment by Giardia lamblia

Giardia lamblia is a protozoan parasite responsible for widespread diarrheal disease in humans and animals. Attachment to the host intestinal mucosa is necessary for establishing infection, but how Giardia performs this attachment is poorly understood. This work illuminates two aspects of Giardia attachment: a molecular analysis to identify microfilament-associated proteins and a mechanistic analysis to examine the dynamics of parasite attachment and detachment.The microfilament system plays an important role in attachment, yet no known microfilament protein except actin in Giardia has been identified. We developed an actin co-sedimentation/mass spectrometry assay using human beta-actin to identify possible microfilament-associated proteins in Giardia. We found that only Giardia actin and two proteins in the alpha-giardin family (alpha-1 giardin and alpha-7.3 giardin) were specifically enriched in F-actin pellets (P<0.2). The specific co-existing of Giardia actin and F-actin suggests that the parasite and human actins can co-polymerize, an interesting finding for such divergent species. The two alpha giardins identified in this study belong to annexin family and localized to the plasma membrane, where actin also localizes, supporting an in vivo role for the observed in vitro interactions.The dynamic changes of the relative topology of substrate and parasite have never been observed and would provide important evidence to understand the attachment mechanism. We used total internal reflection fluorescence (TIRF) microscopy to observe parasites surface-labeled with Alexa-488 conjugated wheat germ agglutinin attach to a glass substrate. We found that the bare zone (a cytoplasmic protrusion through the structurally-unsupported center of the ventral disk), the lateral crest at the periphery of the ventral disk, and the lateral shields at the posterior end of the ventral disk are in closest apposition to the substrate, with the bare zone showing most the dramatic changes during attachment and detachment. In addition, observations of the dynamics of fluorescent microspheres indicated the presence of fluid flow under the surface of the ventral disk of attached parasites. These data provide support for a negative pressure model of attachment.Together these studies advance our understanding of both the machinery of attachment and the biomechanical properties of attachment and point to new directions for chemotherapeutic research.