A Candida glabrata calcineurin mutant exhibited increased susceptibility to both azole antifungal and cell wall-damaging agents and was also attenuated in virulence. Although a mutant lacking the downstream transcription factor Crz1 displayed a cell wall-associated phenotype intermediate to that of the calcineurin mutant and was modestly attenuated in virulence, it did not show increased azole susceptibility. These results suggest that calcineurin regulates both Crz1-dependent and -independent pathways depending on the type of stress.

Proper protein folding in the endoplasmic reticulum (ER) is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR), is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata. The majority of secretory and transmembrane proteins are structurally matured in the endoplasmic reticulum (ER). The accumulation of misfolded proteins in the ER (ER stress) activates the ER-resident stress transducer Ire1, which has two distinct outputs: the unfolded protein response (UPR) and regulated Ire1-dependent decay (RIDD). The UPR is a transcriptional response to increase the protein folding capacity of the ER. RIDD induces degradation of ER-localized mRNAs to reduce the ER load. To date, the UPR has been believed to be an evolutionarily conserved pathway in almost all eukaryotic species, while RIDD has been found only in metazoans. Recent studies in several pathogenic fungi revealed that the UPR is implicated in antifungal resistance and virulence, and thus it has attracted attention as a therapeutic target. Here, we demonstrate that the important fungal pathogen Candida glabrata has lost the canonical UPR, but instead possesses the RIDD pathway and is relatively tolerant to ER stress. The transcriptional response to ER stress was dependent mainly on calcium signaling mediated by the protein phosphatase calcineurin in C. glabrata. Our results provide novel insights into ER quality control mechanisms and are useful for understanding evolutionary biology and the development of antifungal agents targeting the UPR.

The novel arylamidine T-2307 exhibits broad-spectrum in vitro and in vivo antifungal activities against clinically significant pathogens. Previous studies have shown that T-2307 accumulates in yeast cells via a specific polyamine transporter and disrupts yeast mitochondrial membrane potential. Further, it has little effect on rat liver mitochondrial function.

The Slt2 mitogen-activated protein kinase pathway plays a major role in maintaining fungal cell wall integrity. In this study, we investigated the ef

Background: Development of an effective antiviral drug for Coronavirus Disease 2019 (COVID-19) is a global health priority. Although several candidate drugs have been identified through in vitro and in vivo models, consistent and compelling evidence from clinical studies is limited. The lack of evidence from clinical trials may stem in part from the imperfect design of the trials. We investigated how clinical trials for antivirals need to be designed, especially focusing on the sample size in randomized controlled trials. Methods and findings: A modeling study was conducted to help understand the reasons behind inconsistent clinical trial findings and to design better clinical trials. We first analyzed longitudinal viral load data for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) without antiviral treatment by use of a within-host virus dynamics model. The fitted viral load was categorized into 3 different groups by a clustering approach. Comparison of the estimated parameters showed that the 3 distinct groups were characterized by different virus decay rates (p-value < 0.001). The mean decay rates were 1.17 d⁻¹ (95% CI: 1.06 to 1.27 d⁻¹), 0.777 d⁻¹ (0.716 to 0.838 d⁻¹), and 0.450 d⁻¹ (0.378 to 0.522 d⁻¹) for the 3 groups, respectively. Such heterogeneity in virus dynamics could be a confounding variable if it is associated with treatment allocation in compassionate use programs (i.e., observational studies). Subsequently, we mimicked randomized controlled trials of antivirals by simulation. An antiviral effect causing a 95% to 99% reduction in viral replication was added to the model. To be realistic, we assumed that randomization and treatment are initiated with some time lag after symptom onset. Using the duration of virus shedding as an outcome, the sample size to detect a statistically significant mean difference between the treatment and placebo groups (1:1 allocation) was 13,603 and 11,670 (when the antiviral effect was 95% and 99%, respectively) per group if all patients are enrolled regardless of timing of randomization. The sample size was reduced to 584 and 458 (when the antiviral effect was 95% and 99%, respectively) if only patients who are treated within 1 day of symptom onset are enrolled. We confirmed the sample size was similarly reduced when using cumulative viral load in log scale as an outcome. We used a conventional virus dynamics model, which may not fully reflect the detailed mechanisms of viral dynamics of SARS-CoV-2. The model needs to be calibrated in terms of both parameter settings and model structure, which would yield more reliable sample size calculation. Conclusions: In this study, we found that estimated association in observational studies can be biased due to large heterogeneity in viral dynamics among infected individuals, and statistically significant effect in randomized controlled trials may be difficult to be detected due to small sample size. The sample size can be dramatically reduced by recruiting patients immediately after developing symptoms. We believe this is the first study investigated the study design of clinical trials for antiviral treatment using the viral dynamics model.

Gastrointestinal colonization has been considered as the primary source of candidaemia; however, few established mouse models are available that mimic this infection route. We therefore developed a reproducible mouse model of invasive candidiasis initiated by fungal translocation and compared the virulence of six major pathogenic Candida species. The mice were fed a low-protein diet and then inoculated intragastrically with Candida cells. Oral antibiotics and cyclophosphamide were then administered to facilitate colonization and subsequent dissemination of Candida cells. Mice infected with Candida albicans and Candida tropicalis exhibited higher mortality than mice infected with the other four species. Among the less virulent species, stool titres of Candida glabrata and Candida parapsilosis were higher than those of Candida krusei and Candida guilliermondii. The fungal burdens of C. parapsilosis and C. krusei in the livers and kidneys were significantly greater than those of C. guilliermondii. Histopathologically, C. albicans demonstrated the highest pathogenicity to invade into gut mucosa and liver tissues causing marked necrosis. Overall, this model allowed analysis of the virulence traits of Candida strains in individual mice including colonization in the gut, penetration into intestinal mucosa, invasion into blood vessels, and the subsequent dissemination leading to lethal infections.