HAC1 contributes to stress adaptation and virulence in the emerging fungal pathogen Candida auris

This study demonstrates that the unfolded protein response regulator HAC1 is essential for *Candida auris* stress adaptation and virulence, as its unconventional splicing facilitates survival under ER and heat stress, while its deletion significantly reduces fungal burden and host mortality in infection models.

The Gist

The Big Picture: A New Super-Villain Fungus

Imagine a new kind of "super-villain" has entered the world of infections. This villain is a fungus called Candida auris. Unlike its cousins that have been around for a long time, this one is new, very tough, and resistant to almost all the medicines we usually use to kill it. It's like a burglar who has learned to pick every lock and ignore every alarm system.

Doctors and scientists are worried because this fungus is causing serious infections in hospitals, especially in people with weak immune systems. But here's the problem: we don't fully understand how it survives and attacks the human body. To stop it, we need to find its "Achilles' heel"—the one weak spot we can exploit.

The Detective Work: Finding the Weak Spot

The scientists in this study acted like detectives. They wanted to see what genes (the fungus's instruction manuals) were turned "on" when the fungus was actually inside a mouse, compared to when it was just sitting in a petri dish.

They set up a scenario where they infected mice with the fungus and then looked at the fungus's "brain activity" (its transcriptome) while it was fighting the mouse's immune system. They found a specific gene that was screaming "I'm working hard!" whenever the fungus was inside the host.

They named this gene HAC1.

The "Quality Control" Manager

To understand what HAC1 does, imagine the fungus is a busy factory. Inside this factory, there is a special department called the Endoplasmic Reticulum (ER). This is where the factory builds all its important products (proteins).

Sometimes, the factory gets too hot, or the materials get messy, and the products start to get "misfolded" or broken. This is called ER Stress. If too many broken products pile up, the factory shuts down, and the fungus dies.

HAC1 is the factory's emergency manager.

• The Alarm: When things get messy, a sensor sounds the alarm.
• The Splicing (The Magic Trick): The manager (HAC1) has a secret instruction manual. Normally, the manual has a "stop" sign in the middle that makes the manager inactive. But when the alarm sounds, the factory performs a magical trick called unconventional splicing. It literally cuts out a 287-page chunk of the instruction manual and glues the remaining pages together.
• The Result: Suddenly, the manager becomes active and starts shouting orders to fix the broken products, clean up the mess, and keep the factory running.

The Experiment: What Happens When We Fire the Manager?

The scientists decided to test how important this manager was. They created a version of the fungus where they deleted (fired) the HAC1 manager.

Here is what happened:

1. The Heat Test: They put the fungus in a hot environment (like the human body, which is warmer than the outside world). Without the manager, the factory couldn't handle the heat. The fungus struggled to grow.
2. The Chemical Attack: They exposed the fungus to chemicals that mess up protein folding (like tunicamycin). The fungus without the manager crumbled under the pressure.
3. The Infection Test (The Big Showdown):
   • They infected wax moth larvae (a common test subject) and mice with the "manager-less" fungus.
   • Result: The mice and moths lived much longer! The fungus without HAC1 couldn't survive the heat and stress of the host's body. It was like sending an army into battle without their general; they got confused and defeated quickly.

The Twist: Not All Fungi Are the Same

The scientists also looked at different "clades" (families) of Candida auris from different parts of the world (Asia, Africa, South America, etc.).

They found that while all of them have this manager, they use it differently. Some families (like Clade I and IV) are always keeping the manager slightly active, even when things are calm. Others (like Clade II and III) only wake the manager up when things get really bad. This suggests that different strains of this fungus have evolved slightly different strategies for handling stress.

The Takeaway

This study is a huge step forward because it tells us:

1. HAC1 is essential: This gene is a key weapon for Candida auris to survive inside humans.
2. A New Target: If we can develop a drug that stops this "splicing" trick—preventing the manager from getting activated—we might be able to kill this super-fungus or at least make it much weaker, allowing our immune system to finish the job.

In short: The scientists found the fungus's emergency manager. When they removed him, the fungus couldn't handle the heat or the stress of being inside a human, and it lost the battle. This gives us a new blueprint for how to defeat this dangerous invader.

Technical Summary

1. Problem Statement

Candida auris is a multidrug-resistant, emerging fungal pathogen causing severe nosocomial outbreaks with high mortality rates, particularly in immunocompromised hosts. Despite its global threat, the molecular mechanisms driving its virulence and host adaptation remain poorly understood compared to model pathogens like Candida albicans. Specifically, the role of the Unfolded Protein Response (UPR) pathway—a critical cellular mechanism for managing Endoplasmic Reticulum (ER) stress during infection—in C. auris pathogenesis has not been thoroughly characterized.

2. Methodology

The study employed a multi-faceted approach combining in vivo transcriptomics, genetic engineering, and phenotypic assays across five distinct C. auris clades.

• In Vivo Transcriptome Analysis:
  • An immunosuppressed mouse model (C57BL/6J) was used to establish gastrointestinal colonization and systemic dissemination.
  • RNA was extracted from intestinal contents (colonization) and kidneys (dissemination) at 14 days post-infection and compared against in vitro cultures.
  • RNA-seq was performed to identify genes consistently upregulated during infection.
• Genetic Manipulation (CRISPR-Cas9):
  • A deletion mutant (HAC1Δ) and a complemented strain were constructed in the wild-type strain (AR 0390, Clade I) using an RNP-based CRISPR-Cas9 system.
  • The HAC1 gene was replaced with a nourseothricin resistance marker (NAT1) for deletion and a hygromycin resistance marker (HygR) for complementation.
• Virulence Assays:
  • Galleria mellonella: Larvae were infected to screen for virulence defects.
  • Murine Systemic Infection: Immunosuppressed BALB/c mice were infected intravenously to assess survival and fungal burden (CFU/g) in kidneys, liver, and spleen.
• Stress Sensitivity & Splicing Analysis:
  • Spot assays were conducted on media containing ER stressors (Tunicamycin, DTT), cell wall stressors (Calcofluor white, Congo red), and heat stress (43°C).
  • RT-PCR and sequencing were used to detect unconventional splicing of HAC1 mRNA under stress and non-stress conditions across Clades I–V.

3. Key Contributions

• Identification of HAC1 as a Virulence Factor: The study identifies the UPR transcription factor HAC1 as a critical virulence determinant in C. auris, linking ER stress adaptation directly to host survival.
• Discovery of Unconventional Splicing: The authors characterized the specific splicing mechanism of HAC1 in C. auris, revealing a 287-bp intron removal that spans the annotated stop codon, suggesting the current protein annotation may be incomplete.
• Clade-Specific Variability: The study provides the first comparative analysis of HAC1 splicing dynamics and stress sensitivity across the five major C. auris clades, highlighting significant phenotypic diversity.
• Thermotolerance Link: The research establishes a functional link between HAC1 activity and thermotolerance, a crucial factor for survival in mammalian hosts.

4. Key Results

• Transcriptomic Screening: RNA-seq identified 338 genes commonly upregulated in both colonization and dissemination sites. Among these, HAC1 (annotated as B9J08_00296) was prioritized.
• Virulence Defects:
  • In G. mellonella, the HAC1 deletion mutant showed significantly delayed mortality (7-day survival: 50% vs. 10% for wild-type).
  • In mice, the HAC1 mutant exhibited a >10-fold reduction in fungal burden in the kidneys, liver, and spleen compared to the wild-type.
• Splicing Mechanism:
  • HAC1 mRNA undergoes unconventional splicing of a 287-bp region.
  • Splicing is induced by ER stress (Tunicamycin/DTT) but occurs at a basal level even in unstressed conditions, suggesting constitutive UPR activation.
  • Clade Differences: Clade I and IV strains showed strong splicing bands under both stressed and unstressed conditions. In contrast, Clade II and III strains showed limited or no splicing induction, correlating with higher sensitivity to ER stress.
• Stress Phenotypes:
  • The HAC1 mutant displayed increased sensitivity to Tunicamycin (ER stress) and heat (43°C).
  • Notably, the mutant did not show increased sensitivity to DTT or cell wall stressors (Calcofluor white/Congo red), indicating a specific role in ER stress and thermotolerance rather than general cell wall integrity.
• Protein Implications: The spliced region spans the annotated open reading frame (ORF) boundary, suggesting the active Hac1 protein may be 311 amino acids long, differing from current database predictions.

5. Significance

This study significantly advances the understanding of C. auris pathogenesis by:

1. Validating the UPR Pathway: Confirming that the Hac1-mediated UPR is essential for C. auris to survive the hostile environment of a mammalian host, specifically by managing ER stress and heat shock.
2. Highlighting Evolutionary Diversity: Demonstrating that stress response mechanisms are not uniform across C. auris clades, which may explain variations in virulence and antifungal susceptibility observed in clinical settings.
3. Therapeutic Potential: Identifying HAC1 and the UPR pathway as potential targets for novel antifungal strategies. Disrupting this pathway could sensitize C. auris to host defenses and thermal stress, offering a new avenue for treating multidrug-resistant infections.
4. Genomic Correction: Providing evidence that the annotated HAC1 ORF in C. auris requires re-evaluation to accurately reflect the functional protein structure.