Reuwsaat, J. C. V. | Search bioRxiv

bioRxiv2020

Calcium binding protein Ncs1 is calcineurin-regulated in Cryptococcus neoformans and essential for cell division and virulence

Intracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signalling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+-binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and non-stress conditions, as the ncs1{Delta} mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level that correlates with increased expression of the Ca2+ transporter genes, CCH1 and MID1. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1{Delta} mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1{Delta} and cam1{Delta}) and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1-deficency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data shows that Ncs1 plays distinct roles in Ca2+ sensing in C. neoformans despite widespread functional conservation of Ncs1 and other regulators of Ca2+ homeostasis. ImportanceCryptococcus neoformans is the major cause of fungal meningitis in HIV infected patients. Several studies have highlighted the important contribution of Ca2+ signalling and homeostasis to the virulence of C. neoformans. Here, we identify the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signalling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.

microbiology↗

bioRxiv2020

The transcription factor Pdr802 regulates Cryptococcus neoformans Titan cell production and pathogenicity

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1 and Liv3; the transcription factors Stb4, Zfc3 and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity. IMPORTANCEThe pathogenic yeast Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment. Here we describe the role of the transcription factor Pdr802 in the response to host conditions and its impact on C. neoformans virulence. We identified direct targets of Pdr802 and also discovered that it regulates cellular features that influence movement of this pathogen from the lung to the brain, where it causes fatal disease. These findings advance our understanding of a serious disease.

microbiology↗