Internalization of the host alkaline pH signal in a fungal pathogen

Abstract

The ability for cells to internalize extracellular cues allows them to adapt to novel and stressful environments. This adaptability is especially important for microbial pathogens that must sense and respond to drastic changes when encountering the human host. Cryptococcus neoformans is an environmental fungus and opportunistic pathogen that naturally lives in slightly acidic reservoirs, but must adapt to the relative increase in alkalinity in the human host in order to effectively cause disease. The fungal-specific Rim alkaline response signaling pathway effectively converts this extracellular signal into an adaptive cellular response allowing the pathogen to survive in its new environment. The newly identified Rra1 protein, the most upstream component of the C. neoformans Rim pathway, is an essential component of this alkaline response. Previous work connected Rra1-mediated signaling to the dynamics of the plasma membrane. Here we identify the specific mechanisms of Rim pathway signaling through detailed studies of the activation of the Rra1 protein. Specifically, we observe that the Rra1 protein is internalized and recycled in a pH-dependent manner, and that this dynamic pattern of localization further depends on specific residues in its C-terminal tail, clathrin-mediated endocytosis, and the integrity of the plasma membrane. The data presented here continue to unravel the complex and intricate processes of pH-sensing in a relevant human fungal pathogen. These studies will further elucidate general mechanisms by which cells respond to and internalize extracellular stress signals.

Conserved and Divergent Features of pH Sensing in Major Fungal Pathogens

“…This endocytosis of CnRra1 is clathrin-dependent, whereby clathrin coating of CnRra1 vesicles results in the recruitment of ESCRT complexes and downstream-acting CnRim pathway components. Pitstop-2-mediated inhibition of clathrin-dependent endocytosis results in a decrease in Rim101 nuclear localisation [37]. This indicates that clathrin-mediated endocytosis of CnRra1 is essential for the activation of CnRim101.…”

“…In C. neoformans, CnRra1 is localised to the PM, in a manner dependent on (i) integrity of the CnRra1 C-terminus [37] and (ii) extracellular pH (2). GFP-tagged truncated CnRra1 (CnRra1-296 T-GFP), which lacks the majority of the C-terminus, but retains a highly charged region, immediately downstream to the final TMD, is functional and exhibits similar localisation patterns to the full-length version of CnRra1 at both pH 4 and pH 8 with punctate structures forming at the cell surface in lower pH conditions and an increase in endomembrane staining at pH 8.…”

“…In response to environmental alkalinisation, CnRra1 localises first to endocytic vesicles, then to endomembranes such as the perinuclear endoplasmic reticulum or intracellular vesicles. CnRra1 is maintained within membranes via CnNap1 (nucleasome adaptor protein 1) [37]. Acidification of previously alkaline environments results in the recycling of CnRra1 from internal membranes to punctate PM loci; this recycling also occurs, following the successful activation of CnRim101.…”