an-optimized-protocol-for-candida-albicans-infection-schmidtea

An Optimized Protocol for <em>Candida albicans</em> Infection in <em>Schmidtea mediterranea</em> to Study Fungal Pathogenesis and Host Defense

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes most humans. Although typically a benign commensal, dysbiosis ...

UNIVERSITY OF CALIFORNIA, MERCED

The human fungal pathogen Candida albicans is constantly exposed to environmental challenges impacting the cell wall. Signaling pathways coordinate stress adaptation and are essential for commensalism and virulence. The transcription factors Sko1, Cas5, and Rlm1 control the response to cell wall stress caused by the antifungal drug caspofungin. Here, we expand the Sko1 and Rlm1 transcriptional circuit and demonstrate that Rlm1 activates Sko1 cell wall stress signaling. Caspofungin-induced transcription of SKO1 and several Sko1-dependent cell wall integrity genes are attenuated in an rlm1Δ/Δ mutant strain when compared to the treated wild-type strain but not in a cas5Δ/Δ mutant strain. Genome-wide chromatin immunoprecipitation (ChIP-seq) results revealed numerous Sko1 and Rlm1 directly bound target genes in the presence of caspofungin that were undetected in previous gene expression studies. Notable targets include genes involved in cell wall integrity, osmolarity, and cellular aggregation, as well as several uncharacterized genes. Interestingly, we found that Rlm1 does not bind to the upstream intergenic region of SKO1 in the presence of caspofungin, indicating that Rlm1 indirectly controls caspofungin-induced SKO1 transcription. In addition, we discovered that caspofungin-induced SKO1 transcription occurs through self-activation. Based on our ChIP-seq data, we also discovered an Rlm1 consensus motif unique to C. albicans. For Sko1, we found a consensus motif similar to the known Sko1 motif for Saccharomyces cerevisiae. Growth assays showed that SKO1 overexpression suppressed caspofungin hypersensitivity in an rlm1Δ/Δ mutant strain. In addition, overexpression of the glycerol phosphatase, RHR2, suppressed caspofungin hypersensitivity specifically in a sko1Δ/Δ mutant strain. Our findings link the Sko1 and Rlm1 signaling pathways, identify new biological roles for Sko1 and Rlm1, and highlight the complex dynamics underlying cell wall signaling.

An expanded cell wall damage signaling network is comprised of the transcription factors Rlm1 and Sko1 in Candida albicans.

The human fungal pathogen Candida albicans maintains pathogenic and commensal states primarily through cell wall functions. The echinocandin antifungal drug caspofungin inhibits cell wall synthesis and is widely used in treating disseminated candidiasis. Signaling pathways are critical in coordinating the adaptive response to cell wall damage (CWD). C. albicans executes a robust transcriptional program following caspofungin-induced CWD. A comprehensive analysis of signaling pathways at the transcriptional level facilitates the identification of prospective genes for functional characterization and propels the development of novel antifungal interventions. This review article focuses on the molecular functions and signaling crosstalk of the C. albicans transcription factors Sko1, Rlm1, and Cas5 in caspofungin-induced CWD signaling.

Transcriptional regulation of the caspofungin-induced cell wall damage response in Candida albicans.

Whole-genome sequencing is widely used to investigate population genomic variation in organisms of interest. Assorted tools have been independently developed to call variants from short-read sequencing data aligned to a reference genome, including single nucleotide polymorphisms (SNPs) and structural variations (SVs). We developed SNP-SVant, an integrated, flexible, and computationally efficient bioinformatic workflow that predicts high-confidence SNPs and SVs in organisms without benchmarked variants, which are traditionally used for distinguishing sequencing errors from real variants. In the absence of these benchmarked datasets, we leverage multiple rounds of statistical recalibration to increase the precision of variant prediction. The SNP-SVant workflow is flexible, with user options to tradeoff accuracy for sensitivity. The workflow predicts SNPs and small insertions and deletions using the Genome Analysis ToolKit (GATK) and predicts SVs using the Genome Rearrangement IDentification Software Suite (GRIDSS), and it culminates in variant annotation using custom scripts. A key utility of SNP-SVant is its scalability. Variant calling is a computationally expensive procedure, and thus, SNP-SVant uses a workflow management system with intermediary checkpoint steps to ensure efficient use of resources by minimizing redundant computations and omitting steps where dependent files are available. SNP-SVant also provides metrics to assess the quality of called variants and converts between VCF and aligned FASTA format outputs to ensure compatibility with downstream tools to calculate selection statistics, which are commonplace in population genomics studies. By accounting for both small and large structural variants, users of this workflow can obtain a wide-ranging view of genomic alterations in an organism of interest. Overall, this workflow advances our capabilities in assessing the functional consequences of different types of genomic alterations, ultimately improving our ability to associate genotypes with phenotypes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Predicting single nucleotide polymorphisms and structural variations Support Protocol 1: Downloading publicly available sequencing data Support Protocol 2: Visualizing variant loci using Integrated Genome Viewer Support Protocol 3: Converting between VCF and aligned FASTA formats.

SNP-SVant: A Computational Workflow to Predict and Annotate Genomic Variants in Organisms Lacking Benchmarked Variants.

The Hxk1 protein of  phosphorylates N-acetylglucosamine (GlcNAc) which is necessary for various cellular functions, including energy production and chitin synthesis. Further, this protein also regulates morphogenesis independently of its role in GlcNAc catabolism. When  is deleted, cells are hyperfilamentous on serum-containing medium. Furthermore, Hxk1 translocates to the nucleus in the presence of glucose. To gain a broad understanding of the effect of Hxk1 on gene expression in , we performed genome-wide transcriptional profiling of the  mutant strain by RNA-Seq. The analysis of these RNA-Seq data showed that Hxk1 affects gene expression in both a carbon source-dependent and -independent manner. However, the effect on gene expression occurs via an indirect mechanism, as genome-wide CUT&RUN binding experiments demonstrated that Hxk1 does not bind to the upstream intergenic regions of the differentially expressed genes. Deletion of  not only resulted in differences in gene expression of genes present in the GlcNAc and galactose regulons, but also in glucose transporter genes, including . Hxk1 also negatively influences the expression of virulence-associated genes, including , , and . Consequently, an  mutant strain showed higher toxicity toward gut epithelial cells compared to the WT strain. Furthermore, the  mutant strain had higher expression levels of  and  and showed higher resistance toward HO. These findings highlight the multiple functions of Hxk1 in different cellular processes.IMPORTANCE is a fungus that lives in the human body but does not cause any harm in healthy individuals. However, when the immune system is weakened,  can spread via the bloodstream all over the body and can lead to severe illness and even death. To infect the human body, multiple proteins hold distinct functions. Hxk1 is one of these proteins. This protein is involved in N-acetylglucosamine (GlcNAc) phosphorylation, as well as hyphae formation and glucose transport. To obtain a complete view of the processes regulated by Hxk1, we performed an RNA-Seq experiment. These data revealed that Hxk1 influences the regulation of genes involved in metabolic and virulence-related processes, such as GlcNAc metabolism, sterol metabolism, and oxidative stress resistance. These findings are important to better understand how  adapted itself to infect the host.

 Hxk1 influences expression of metabolic- and virulence-related genes.

Insects ally with microbial symbionts for a diversity of services. The range of these interactions is wide, spanning from beneficial to pathogenic and facultative to obligate. In many cases, such insect-microbial interactions veer towards mutual dependency with integrated physiologies. This evolutionary outcome is relatively common in insects that depend on microbes to fill gaps in their nutritional ecologies (e.g. plant-sap feeding). However, the initiation and transition towards such dependent symbiotic interactions are difficult to observe in nature. Identifying these events can provide key insights into the origins and evolutionary processes that shape symbiotic interactions. Here, we report on a novel interaction between a leafhopper (Typhlocybinae: Empoasca mexicana) and a bacterium, Symbiopectobacterium purcellii MEX strain (S-MEX). To characterize this symbiont, we assembled and annotated its complete genome. We compared its content and structure to the genomes of other Symbiopectobacterium. The S-MEX genome is unique among members of this genus. It is the largest yet sequenced at 5.3 Mb, encoding 6,838 genes (∼25% more than other strains). S-MEX's genome has significantly expanded due to the proliferation of insertion sequences and 2,723 identifiable pseudogenes-processes generally seen as accelerators of genome reduction and emerging host dependence. S-MEX and other Symbiopectobacterium strains have a core set of 818 genes shared in >90% of strains, of which S-MEX has uniquely lost 36 genes. Taken together, we hypothesize that due to expansion of IS elements, extensive pseudogenization, and loss of genes in important free-living functions, S-MEX is in the early stages of establishing a host-dependent symbiosis.

Characterizing a Novel Symbiopectobacterium purcellii MEX Strain at the Early Stages of Establishing a Symbiotic Relationship.

Antifungal tolerance can promote the emergence of resistance yet often incurs fitness costs for fungal pathogens. How tolerant populations compensate for these deficits and how they may be therapeutically targeted remain poorly understood. Here, we investigate four sequential  isolates recovered from a patient with persistent candidemia and failure of micafungin therapy. The infection was ultimately cleared with liposomal amphotericin B (LAMB). Whole-genome sequencing (WGS) confirmed clonal relatedness and the absence of known resistance mutations. Later isolates displayed marked cell wall remodeling (CWR), characterized by increased mannan and reduced β-glucan content, as revealed by microscopy and solid-state nuclear magnetic resonance. These isolates formed thicker biofilms and displayed enhanced echinocandin tolerance but paradoxically showed increased susceptibility to amphotericin B (AMB)  and during systemic infection in mice. Despite a complex mutational landscape, transcriptomic profiling across planktonic and biofilm growth showed minimal divergence from the earliest isolate. Functionally, evolved isolates suppressed M1 macrophage polarization, dampened proinflammatory cytokine production, survived better during neutrophil interactions, and transiently increased fungal burden . These findings show that host-driven CWR could promote echinocandin tolerance while simultaneously sensitizing  to AMB. Our results suggest that alternating echinocandin and LAMB therapy may effectively eliminate echinocandin-tolerant fungal populations.IMPORTANCEAntifungal tolerance is increasingly recognized as a precursor to resistance, yet its clinical and biological consequences remain poorly defined. By analyzing sequential  isolates from a case of persistent candidemia, we show that cell wall remodeling is associated with echinocandin tolerance, alters host immune interactions, and increases susceptibility to amphotericin B (AMB). These findings reveal how tolerance-associated adaptations shape pathogen fitness during infection and highlight the therapeutic potential of alternating echinocandin and AMB therapy. This work advances our understanding of antifungal tolerance and suggests that exploiting opposing drug susceptibilities may improve treatment outcomes for challenging-to-treat  infections.

Article	Year
An expanded cell wall damage signaling network is comprised of the transcription factors Rlm1 and Sko1 in Candida albicans. PLoS genetics\| PubMed ID: 32639995	2020
Transcriptional regulation of the caspofungin-induced cell wall damage response in Candida albicans. Current genetics\| PubMed ID: 32876716	2020
SNP-SVant: A Computational Workflow to Predict and Annotate Genomic Variants in Organisms Lacking Benchmarked Variants. Current protocols\| PubMed ID: 38717471	2024
Hxk1 influences expression of metabolic- and virulence-related genes. mSphere\| PubMed ID: 40996045	2025
Characterizing a Novel Symbiopectobacterium purcellii MEX Strain at the Early Stages of Establishing a Symbiotic Relationship. Genome biology and evolution\| PubMed ID: 41512111	2026
Profound cell wall remodeling in during systemic infection confers simultaneous tolerance to echinocandins and host immunity. Microbiology spectrum\| PubMed ID: 41685898	2026

Deepika Gunasekaran

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