Unique Evolution of the UPR Pathway with a Novel bZIP Transcription Factor, Hxl1, for Controlling Pathogenicity of Cryptococcus neoformans

Abstract

In eukaryotic cells, the unfolded protein response (UPR) pathway plays a crucial role in cellular homeostasis of the endoplasmic reticulum (ER) during exposure to diverse environmental conditions that cause ER stress. Here we report that the human fungal pathogen Cryptococcus neoformans has evolved a unique UPR pathway composed of an evolutionarily conserved Ire1 protein kinase and a novel bZIP transcription factor encoded by HXL1 (HAC1 and XBP1-Like gene 1). C. neoformans HXL1 encodes a protein lacking sequence homology to any known fungal or mammalian Hac1/Xbp1 protein yet undergoes the UPR-induced unconventional splicing in an Ire1-dependent manner upon exposure to various stresses. The structural organization of HXL1 and its unconventional splicing is widely conserved in C. neoformans strains of divergent serotypes. Notably, both C. neoformans ire1 and hxl1 mutants exhibited extreme growth defects at 37°C and hypersensitivity to ER stress and cell wall destabilization. All of the growth defects of the ire1 mutant were suppressed by the spliced active form of Hxl1, supporting that HXL1 mRNA is a downstream target of Ire1. Interestingly, however, the ire1 and hxl1 mutants showed differences in thermosensitivity, expression patterns for a subset of genes, and capsule synthesis, indicating that Ire1 has both Hxl1-dependent and -independent functions in C. neoformans. Finally, Ire1 and Hxl1 were shown to be critical for virulence of C. neoformans, suggesting UPR signaling as a novel antifungal therapeutic target.

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans

“…As a cytoplasmic molecular chaperone complex, genes encoding a heat shock protein (HSP) Hsp104 (CNAG_07347), Ydj1p/Hsp40 (CNAG_03944), and Ssa1/Hsp70 (CNAG_01750) were upregulated during the temperature upshift. Genes encoding ER-resident molecular chaperones, LHS1 (CNAG_03899) and KAR2 (CNAG_06443), were also upregulated, further supporting our previous finding that the UPR pathway in the ER is activated in response to temperature upshift (Cheon et al 2011). Interestingly, protein refolding within the ER is known to also require the cytoplasmic chaperone Hsp104 (Hänninen et al 1999).…”

“…Due to such reasons, signaling cascades governing thermotolerance in fungi have been intensively investigated toward their exploitation as potential antifungal drug targets (Bahn et al 2007;Bahn and Jung 2013). In C. neoformans, temperature-sensing signaling cascades include the calmodulin/calcineurin (Fox et al 2001;Kraus et al 2005), Ras (Alspaugh et al 2000;Nichols et al 2007), HOG (Bahn et al , 2006, Msi1-like protein (Yang et al 2012), protein kinase C 1 (Pkc1)/ Mpk1 MAPK (Kraus et al 2003;Gerik et al 2005), and unfolded protein response (UPR) (Cheon et al 2011) pathways. Unlike other temperature-sensing signaling components described above, a protein kinase, Sch9, suppresses C. neoformans thermotolerance (Wang et al 2004).…”

The unfolded protein response (UPR) pathway inCryptococcus

“…41 The Cryptococcus Ire1 kinase is highly homologous to the S. cerevisiae Ire1 and to IRE1α and IRE1β in humans. Notably, however, Cryptococcus has a unique bZIP transcription factor encoded by HXL1, which is structurally and phylogenetically distant from yeast Hac1/human XBP1.…”

“…34,42 Upon ER stress, the endonuclease activity of Ire1 removes an unconventional intron, thereby converting unspliced HXL1 mRNA to spliced HXL1 mRNA. 41 Although HXL1 u and HXL1 s mRNAs encode the same bZIP domain at the N-terminus (60 to 125 aa), the HXL1 s mRNA produces the active Hxl1 protein with an extended C-terminal domain (406 aa of Hxl1 u to 426 aa of Hxl1 i ) via frameshift translation of its C-terminal exon region. The Ire1-mediated HXL1 mRNA splicing event is well conserved in different serotypes of Cryptococcus strains, including serotype A C. neoformans var grubii (the H99 strain), serotype D C. neoformans var neoformans (JEC21 and B-3501A strains), and serotype B C. gattii (R265 and WM276 strains).…”

“…The Ire1-mediated HXL1 mRNA splicing event is well conserved in different serotypes of Cryptococcus strains, including serotype A C. neoformans var grubii (the H99 strain), serotype D C. neoformans var neoformans (JEC21 and B-3501A strains), and serotype B C. gattii (R265 and WM276 strains). 41 The target genes regulated by the UPR pathway play critical roles in counteracting ER stress. Transcriptome profiling analysis of the S. cerevisiae UPR pathway defined the "UPR regulon", which includes genes involved in the protein secretion and modification pathway as well as ER-resident chaperones.…”