ABSTRACTCompared to other fungal pathogens,Cryptococcus neoformansis particularly adept at avoiding detection by innate immune cells. To explore fungal cellular features involved in immune avoidance, we characterized cell surface changes of theC. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strainrim101Δ cell wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that therim101Δ strain shows a modest increase in the levels of both cell wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of cell wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing therim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to therim101Δ mutantin vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates theC. neoformanscell wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune cells detectC. neoformansand other fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan.IMPORTANCEInfectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their cell surfaces to mask immunogenic epitopes. We have created a fungal strain with a targeted mutation in a pH response pathway that is unable to properly organize its cell wall, resulting in a dramatic immune reaction during infection. This mutant cell wall is defective in hiding important cell wall components, such as the chito-oligomers chitin and chitosan. By creating a series of cell wall mutants, we demonstrated that the degree of chito-oligomer exposure correlates with the intensity of innate immune cell activation. This activation requires a combination of host receptors to recognize and respond to these infecting microorganisms. Therefore, these experiments explored host-pathogen interactions that determine the degree of the subsequent inflammatory response and the likely outcome of infection.