Abstract
The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.
Highlights
The microbial surface is the first point of contact for interactions with the innate immune system, representing the site at which an infected host might recognize a microbe as a potential pathogen
This recognition is achieved through host pattern recognition receptors (PRRs) that distinguish specific pathogen-associated molecular patterns (PAMPs) on microbial surfaces, directing downstream signaling events that lead to the initiation of an immune response
We further show that this innate immune recognition is dependent on the Card9 and MyD88 adaptor proteins and the cell surface receptors Dectin-1 and TLR2
Summary
The microbial surface is the first point of contact for interactions with the innate immune system, representing the site at which an infected host might recognize a microbe as a potential pathogen. The fungal cell wall is a dynamic structure composed of a complex matrix of polysaccharides including α- and β-glucans, mannoproteins (mannans), and chitin/chitosan. These fungal specific components have been shown by many groups to be recognized by host PRRs including Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) [1,2]. Candida albicans differentially exposes β-glucan in response to diverse host niches, drug treatments, and growth conditions, resulting in varying degrees of Dectin-1-mediated host responses [5,6]
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