The protein kinase Ire1 impacts pathogenicity of Candida albicans by regulating homeostatic adaptation to endoplasmic reticulum stress

The Ire1-mutant strain is severely defective in establishing a successful infection in vivo.

“Furthermore, the Ire1‐mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo.”

Ire1 activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate splicing of HAC1 mRNA.

“Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA.”

An Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits.

“Concordantly, an Ire1‐mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence‐related traits.”

During tunicamycin treatment, HAC1 u mRNA was processed in mkc1Δ/Δ cells similar to wild-type levels.

“During tunicamycin treatment, HAC1 u mRNA was processed in the mkc1Δ/Δ cells similar to wild‐type levels (Figure 4e)”

The hyphal defect in the ire1 DX mutant strain was more pronounced after 24 hr as evident by the presence of a large proportion of yeast cells.

“The hyphal defect in the ire1 DX mutant strain was more pronounced after 24 hr as evident by the presence of a large proportion of yeast cells (Figure 5b).”

The Mkc1 MAPK was constitutively phosphorylated (1.7-fold) in the ire1 DX mutant strain compared to the wild-type strain.

“The Mkc1 MAPK was constitutively phosphorylated (1.7‐fold) in the ire1 DX mutant strain, compared to the wild‐type strain (Figure 4d).”

In the ire1 DX mutant compared to wild type grown in YEPD, 228 genes were downregulated and 362 genes were upregulated with expression fold changes of more than 1.5 and less than -1.5 and p values ≤ .05.

“A total of 228 genes were downregulated and 362 genes were upregulated with expression fold changes of more than 1.5 and less than −1.5 with p values ≤.05 in the ire1 DX mutant strain compared to the wild‐type strain.”

Compromising Ire1 function results in abrogated intron splicing from HAC1 mRNA.

“(b) abrogated intron splicing from HAC1 mRNA”

Compared to wild-type and complemented strain, the ire1 DX mutant strain exhibited considerably increased susceptibility to Congo red, calcofluor white, tunicamycin, DTT, 2-mercaptoethanol, fluconazole and ketoconazole.

“Compared to the wild‐type and complemented strain, the ire1 DX mutant strain exhibited considerably increased susceptibility to the cell wall stressors Congo red and calcofluor white (CFW); the ER stressors tunicamycin, DTT and 2‐mercaptoethanol (β‐ME) and the azole antifungal drugs fluconazole and ketoconazole (Figure 1c).”

The ire1 DX mutant strain exhibits increased susceptibility to compounds interfering with cell membrane integrity (SDS and amphotericin B).

“The increased susceptibility of the ire1 DX mutant strain to these compounds confirmed the requirement of Ire1 in maintaining membrane stability (Figure 4a).”

The ire1 DX mutant was impaired in the same virulence-associated processes.

“Concordantly, the mutant was impaired in these same virulence‐associated processes (Figure 5).”

Levels of spliced HAC1 mRNA in tunicamycin-treated cmp1Δ/Δ cells remained similar to treated wild-type cells.

“We show that the levels of spliced HAC1 mRNA in tunicamycin‐treated cmp1Δ/Δ cells remained similar to the treated wild‐type cells (Figure S4)”

In wild-type C. albicans cells grown in medium containing inositol for 5 hr, Ire1-dependent splicing of HAC1 mRNA occurs.

“This experiment shows the occurrence of Ire1‐dependent splicing of HAC1 mRNA in wild‐type cells grown in medium containing inositol for 5 hr”

Genes associated with cell wall biogenesis and virulence traits such as hyphal morphogenesis and biofilm formation (NAG3, ERG2, FET34, FTR1, YWP1 and RBE1) featured prominently in the top 50 downregulated genes in the ire1 DX mutant strain.

“genes associated with these functional categories (NAG3, ERG2, FET34 and FTR1, YWP1 and RBE1) also featured prominently in the top 50 downregulated genes.”

Transcriptional profiling analysis revealed downregulation of genes associated with maintaining iron homeostasis (FET34, RBT5, FET3 and FRE10) in the ire1 DX mutant strain.

“Transcriptional profiling analysis revealed downregulation of genes associated with maintaining iron homeostasis (FET34, RBT5, FET3 and FRE10), pointing towards the remodelling of iron‐dependent metabolic pathways in the ire1 DX mutant strain.”

The inability of the ire1 DX mutant strain to grow on ER stressors and process HAC1 u mRNA was rescued by intact IRE1 but not by IRE1-KD or IRE1-ND mutant versions of Ire1.

“The inability of the ire1 DX mutant strain to grow on ER stressors and process HAC1 u mRNA was rescued by intact IRE1 but not by the IRE1‐KD (kinase dead) or IRE1‐ND (nuclease dead)‐mutant versions of Ire1”

The ire1 DX mutant strain exhibited changes in expression of genes encoding proteins that promote virulence-associated processes such as iron assimilation, cell wall biogenesis, hyphal morphogenesis and biofilm formation.

“Furthermore, the mutant strain also exhibited changes in the expression of genes encoding proteins that promote virulence‐associated processes such as iron assimilation, cell wall biogenesis, hyphal morphogenesis and biofilm formation (Figure 2).”

Tunicamycin treatment resulted in significantly enhanced levels of phosphorylated Mkc1 in the ire1 DX mutant strain (2.6-fold) compared to the treated wild-type strain.

“Tunicamycin treatment resulted in significantly enhanced levels of phosphorylated Mkc1 in the ire1 DX mutant strain (2.6‐fold), compared to the treated wild‐type strain (Figure 4d).”

HAC1 splicing was more pronounced at 5 hr in medium lacking inositol than in medium containing inositol.

“In contrast, HAC1 splicing was more pronounced at 5 hr in medium lacking inositol (Figure 4b)”

Compromised expression of Ire1 did not result in changes in cell wall architecture as assessed by transmission electron microscopy and cell wall composition analysis.

“Furthermore, compromised expression of Ire1 did not result in changes in cell wall architecture as assessed by transmission electron microscopy and cell wall composition analysis (data not shown).”

FET34 and FTR1 are among the highest downregulated genes in the ire1 DX mutant strain.

“FET34 and FTR1 are among the highest downregulated genes that are shown to be activated in iron‐limiting conditions.”

HAC1 u mRNA was not processed at any tested time points (1, 3, and 5 hr) in fluconazole- and CFW-treated wild-type cells.

“HAC1 u mRNA was not processed at any of the tested time points in fluconazole‐ and CFW‐treated wild‐type cells (Figure 4f,g and Figure S3).”

The biofilm-forming abilities of the ire1 DX mutant strain was reduced by ~1.5-fold relative to the wild-type strain.

“The biofilm‐forming abilities of the ire1 DX mutant strain was reduced by ~1.5‐fold relative to the wild‐type strain (Figure 5d).”

Compromising Ire1 function results in growth defects in the presence of ER stress-inducing agents (tunicamycin and DTT).

“compromising Ire1 function results in (a) growth defects in the presence of ER stress‐inducing agents (tunicamycin and DTT)”

The ire1 DX mutant strain exhibits increased susceptibility to cell wall-damaging agents.

“Increased susceptibility of the ire1 DX mutant strain to cell wall‐damaging agents (Figure 1c)”

Wild-type C. albicans cells treated with tunicamycin, CFW, fluconazole or inositol-depleted medium for 1 and 5 hr did not exhibit increased IRE1 mRNA levels compared to untreated wild type.

“Wild‐type C. albicans cells treated with these stressors for 1 and 5 hr did not exhibit an increase in IRE1 mRNA levels, compared to the untreated wild type (Figure S2B).”

Compromising Ire1 function results in decreased expression of Hac1-dependent ER stress response genes.

“(c) decreased expression of Hac1‐dependent ER stress response genes (Figures 1c and 3a–c)).”

A similar hyphal defect in the ire1 DX mutant strain was also observed in all media tested.

“A similar hyphal defect in the ire1 DX mutant strain was also observed in all media tested (Figure S5)”

Fluconazole treatment of wild-type cells did not necessitate processing of HAC1 u mRNA over time.

“On the contrary, we observed that fluconazole treatment of the wild‐type cells did not necessitate the processing of HAC1 u mRNA over time (Figure 4f).”

C. albicans Ire1 responded to membrane aberrations induced by inositol depletion by promoting splicing of HAC1 mRNA.

“In line with this idea, C. albicans Ire1 also responded to membrane abberations, induced by inositol depletion, by promoting the splicing of HAC1 mRNA (Figure 4b).”

Interfering with cell wall integrity (calcofluor white treatment) that indirectly affects membrane homeostasis also did not necessitate processing of HAC1 u mRNA.

“Likewise, interfering with cell wall integrity (calcofluor white treatment) that indirectly affects membrane homeostasis (Promlek et al., 2011) also did not necessitate the processing of HAC1 u mRNA (Figure 4g).”

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Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis.

“Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling.”

C. albicans Ire1 functions to regulate traits that are essential for virulence.

“Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.”

Candida albicans relies on the ER-resident protein Ire1 for sensing ER stress and activating the unfolded protein response.

“Here, we show that Candida albicans relies on an ER‐resident protein, inositol‐requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR.”

The UPR is involved in a broad range of cellular processes related to secretory homeostasis, as inferred from transcriptome analysis.

“and is also involved in a broad range of cellular processes related to secretory homeostasis, which can be inferred from our transcriptome analysis (Figure 2a).”

Ire1 is implicated in maintaining cell wall integrity via its role in maintaining secretory homeostasis in C. albicans.

“and implicates Ire1 in maintaining cell wall integrity via its role in maintaining secretory homeostasis in C. albicans.”

Ire1 is classified as an essential gene in C. albicans based on a prior study that was unable to obtain a null mutant of this gene.

“Ire1 is classified as an essential gene based on a prior study that was unable to obtain a null mutant of this gene in C. albicans (Blankenship, Fanning, Hamaker, & Mitchell, 2010).”

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Ire1 regulates C. albicans pathogenesis by integrating expression of virulence-related traits with ER-dependent maintenance of secretory homeostasis.

“We posit that Ire1 regulates C. albicans pathogenesis by integrating the expression of virulence‐related traits with ER‐dependent maintenance of secretory homeostasis.”

Both kinase and nuclease activities are indispensible in facilitating the capacity of Ire1 to counter ER stress.

“indicating that both the kinase and nuclease activities are indispensible in facilitating the capacity of this protein kinase to counter ER stress (Figure 3d,e).”

C. albicans relies on Ire1-mediated stress responses for its pathogenesis.

“In this study, we show that C. albicans also relies on Ire1‐mediated stress responses for its pathogenesis, thus expanding the link between Ire1‐dependent stress responsive pathways and fungal pathogenesis to C. albicans.”

Functional Ire1 may have a role in facilitating adaptation of C. albicans to low iron stress.

“indicating that functional Ire1 may have a role in facilitating adaptation of C. albicans to low iron stress.”

Ire1-dependent UPR is essential for circumventing cell membrane and cell wall stress in C. albicans.

“(c) Ire1‐dependent UPR is essential for circumventing cell membrane and cell wall stress in C. albicans.”

Ire1 is required for normal biofilm development both in vitro and in vivo in C. albicans.

“These findings demonstrate that Ire1 is required for normal biofilm development both in vitro and in vivo in C. albicans.”

The calcineurin pathway does not directly regulate processing of HAC1 u mRNA in C. albicans.

“indicating that, similar to the Mkc1 MAPK pathway, the calcineurin pathway does not directly regulate the processing of the HAC1 u mRNA in this pathogenic fungus.”

Ire1 is required for maintaining membrane stability in C. albicans.

“The increased susceptibility of the ire1 DX mutant strain to these compounds confirmed the requirement of Ire1 in maintaining membrane stability (Figure 4a).”

The core domain structure of Ire1 is conserved among C. albicans, S. cerevisiae, C. glabrata, A. fumigatus and C. neoformans.

“Sequence alignment results suggest that the core domain structure of Ire1 is conserved among these fungal species.”

In order to circumvent iron-starved environments, C. albicans activates expression of essential iron regulon genes that facilitate iron acquisition from the host.

“In order to circumvent iron‐starved environments, C. albicans activates the expression of essential iron regulon genes (Chen & Noble, 2012) that facilitate iron acquisition from the host (Fourie, Kuloyo, Mochochoko, Albertyn, & Pohl, 2018).”

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Stress-dependent activation of Ire1 may largely be dependent on post-transcriptional or translational modifications affecting its oligomeric status.

“This indicates that stress‐dependent activation of Ire1 may largely be dependent on post‐transcriptional/translational modifications, in turn affecting its oligomeric status, which is a prerequisite for stimulating its kinase and RNase activities.”

Growth for long durations in inositol-containing medium may induce ER stress leading to activation of the UPR.

“indicating that growth for long durations in this medium may be inducing ER stress leading to activation of the UPR.”

These results place Ire1 as the link between protein quality control and membrane homeostasis for maintenance of cell wall/membrane integrity in C. albicans.

“Overall, these results place Ire1 as the link between protein quality control and membrane homeostasis for the maintainance of cell wall/membrane integrity in C. albicans.”

All of the virulence traits (iron adaptation, hyphal morphogenesis, biofilm formation) are supported by an intact Ire1-dependent secretory pathway.

“All of these virulence traits are supported by an intact Ire1‐dependent secretory pathway (Cheon et al., 2011; Feng et al., 2011; Jung et al., 2013).”

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These results highlight the reliance of C. albicans on the canonical Ire1-Hac1 UPR pathway for ER stress response and homeostasis.

“Overall, these results highlight the reliance of C. albicans on the canonical Ire1‐Hac1 UPR pathway for ER stress response and homeostasis.”

A healthy working UPR is required for cell wall maintenance in C. albicans.

“Increased susceptibility of the ire1 DX mutant strain to cell wall‐damaging agents (Figure 1c) also pointed to the requirement of a healthy working UPR for cell wall maintenance in C. albicans.”

Ire1 is essential for C. albicans growth in the presence of different stressors.

“We infer that Ire1 is essential for C. albicans growth in the presence of different stressors.”

Ire1 impacts hyphal morphogenesis in C. albicans.

“confirming the impact of Ire1 on hyphal morphogenesis in C. albicans.”

The Mkc1 MAPK pathway does not directly regulate splicing of HAC1 mRNA during ER stress.

“suggesting that the Mkc1 MAPK pathway does not directly regulate the splicing of HAC1 mRNA during ER stress.”

Several important C. albicans virulence traits are intertwined with the ability of Ire1 to support secretory homeostasis in C. albicans.

“Taken together, these results indicate that several important C. albicans virulence traits are intertwined with the ability of Ire1 to support secretory homeostasis in C. albicans.”

The inability of the ire1 DX mutant to sustain growth in the presence of cell wall stressors is primarily a consequence of impaired secretory homeostasis due to ER stress.

“and the inability of the mutant to sustain growth in the presence of cell wall stressors is primarily a consequence of impaired secretory homeostasis due to ER stress.”

Defective secretory homeostasis is the basis for increased susceptibility to cell wall stressors in the ire1 DX mutant strain.

“Collectively, these results suggest that defective secretory homeostasis is the basis for increased susceptibility to cell wall stressors in the ire1 DX mutant strain”

ER stress-induced loss of cell viability is not due to an intrinsic cell wall defect in the ire1 DX mutant strain.

“This finding indicates that ER stress‐induced loss of cell viability is not due to an intrinsic cell wall defect in the ire1 DX mutant strain”

During infection in vivo, C. albicans routinely encounters iron deficiency due to iron sequestration by the host.

“During infection in vivo, C. albicans routinely encounters iron deficiency due to iron sequestration by the host (Ramanan, 2000).”

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It is likely that Ire1 impacts azole susceptibility via its ability to sense aberrations in membrane homeostasis.

“Thus, it is likely that Ire1 impacts azole susceptibility via its ability to sense aberrations in membrane homeostasis.”

Our data show that Ire1 responds to accumulation of misfolded proteins as well as membrane stress to activate the UPR in C. albicans.

“Nevertheless, our data show that Ire1 responds to the accumulation of misfolded proteins as well as membrane stress to activate the UPR in C. albicans.”

Coping with ER stress also requires activation of complementary signalling pathways such as Mkc1 MAPK and calcineurin pathways that operate in parallel with the Ire1-dependent UPR pathway in a manner that does not influence processing of HAC1 u mRNA in C. albicans.

“Coping with ER stress also requires activation of complementary signalling pathways such as the Mkc1 MAPK and calicneurin pathways that operate in parallel with the Ire1‐dependent UPR pathway in a manner that does not influence the processing of HAC1 u mRNA in C. albicans (Figure 4e and Figures S4 and S6).”

The ability of Ire1 to jointly respond to both membrane and protein homeostasis enables Ire1 to activate the UPR in response to multiple stressors in C. albicans.

“The ability of Ire1 to jointly respond to both membrane and protein homeostasis enables Ire1 to activate the UPR in response to multiple stressors in C. albicans.”

Our data demonstrate that accumulation of misfolded proteins during tunicamycin-induced acute ER stress induces the UPR by promoting splicing of HAC1 u mRNA in an Ire1-dependent manner in C. albicans.

“In conclusion, our data demonstrate that the accumulation of misfolded proteins during tunicamycin‐induced acute ER stress induces the UPR by promoting the splicing of HAC1 u mRNA in an Ire1‐dependent manner in C. albicans (Figure 6).”

Ire1-dependent activation of the UPR occurs not only upon accumulation of misfolded proteins but also during lipid bilayer stress.

“Ire1‐dependent activation of the UPR occurs not only upon the accumulation of misfolded proteins but also during lipid bilayer stress.”

Our data support the idea that Ire1 plays a role in mediating multiple cellular processes via its ability to maintain secretory homeostasis in C. albicans.

“Thus, our data support the idea that Ire1 plays a role in mediating multiple cellular processes via its ability to maintain secretory homeostasis in C. albicans.”

Our study reveals a link between lipid homeostasis and the Ire1-dependent UPR pathway in C. albicans.

“Thus, our study reveals a link between lipid homeostasis and the Ire1‐dependent UPR pathway in C. albicans.”