Using COVID-19 as a teaching tool in a time of remote learning: A workflow for bioinformatic approaches to identifying candidates for therapeutic and vaccine development.

The COVID-19 pandemic has led to an urgent need for engaging computational alternatives to traditional laboratory exercises. Here we introduce a customizable and flexible workflow, designed with the SARS CoV-2 virus that causes COVID-19 in mind, as a means of reinforcing fundamental biology concepts using bioinformatics approaches. This workflow is accessible to a wide range of students in life science majors regardless of their prior bioinformatics knowledge, and all software is freely available, thus eliminating potential cost barriers. Using the workflow can thus provide a diverse group of students the opportunity to conduct inquiry-driven research. Here we demonstrate the utility of this workflow and outline the logical steps involved in the identification of therapeutic or vaccine targets against SARS CoV-2. We also provide an example of how the workflow may be adapted to other infectious microbes. Overall, our workflow anchors student understanding of viral biology and genomics and allows students to develop valuable bioinformatics expertise as well as to hone critical thinking and problem-solving skills, while also creating an opportunity to better understand emerging information surrounding the COVID-19 pandemic.

Caenorhabditis elegans as a Model Host to Monitor the Candida Infection Processes.

C. elegans has several advantages as an experimental host for the study of infectious diseases. Worms are easily maintained and propagated on bacterial lawns. The worms can be frozen for long term storage and still maintain viability years later. Their short generation time and large brood size of thousands of worms grown on a single petri dish, makes it relatively easy to maintain at a low cost. The typical wild type adult worm grows to approximately 1.5 mm in length and are transparent, allowing for the identification of several internal organs using an affordable dissecting microscope. A large collection of loss of function mutant strains are readily available from the C. elegans genetic stock center, making targeted genetic studies in the nematode possible. Here we describe ways in which this facile model host has been used to study Candida albicans, an opportunistic fungal pathogen that poses a serious public health threat.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans.

Almost all humans are colonized with Candida albicans However, in immunocompromised individuals, this benign commensal organism becomes a serious, life-threatening pathogen. Here, we describe and analyze the regulatory networks that modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous sequence analysis of clinical C. albicans isolates from immunocompromised patients indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen species (ROS), suggesting they help neutralize the host-derived ROS produced by phagocytes, as well as establish a sustained infection in vivo Transcriptomic analysis of mutants under resting conditions where cells were not experiencing oxidative stress revealed a large network that control macro and micronutrient homeostasis, which likely contributes to overall pathogen fitness in host niches. Under oxidative stress, both transcription factors regulate a separate set of genes involved in detoxification of ROS and down-regulating ribosome biogenesis. ChIP-seq analysis, which reveals vastly different binding partners for each transcription factor (TF) before and after oxidative stress, further confirms these results. Furthermore, the absence of a dominant binding motif likely facilitates their mobility, and supports the notion that they represent a recent expansion of the ZCF family in the pathogenic Candida species. Our analyses provide a framework for understanding new aspects of the interface between C. albicans and host defense response, and extends our understanding of how complex cell behaviors are linked to the evolution of TFs.

The evolution of drug resistance in clinical isolates of Candida albicans.

Candida albicans is both a member of the healthy human microbiome and a major pathogen in immunocompromised individuals. Infections are typically treated with azole inhibitors of ergosterol biosynthesis often leading to drug resistance. Studies in clinical isolates have implicated multiple mechanisms in resistance, but have focused on large-scale aberrations or candidate genes, and do not comprehensively chart the genetic basis of adaptation. Here, we leveraged next-generation sequencing to analyze 43 isolates from 11 oral candidiasis patients. We detected newly selected mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss-of-heterozygosity (LOH) events. LOH events were commonly associated with acquired resistance, and SNPs in 240 genes may be related to host adaptation. Conversely, most aneuploidies were transient and did not correlate with drug resistance. Our analysis also shows that isolates also varied in adherence, filamentation, and virulence. Our work reveals new molecular mechanisms underlying the evolution of drug resistance and host adaptation.

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis.

Infection by pathogenic fungi, such as Candida albicans, begins with adhesion to host cells or implanted medical devices followed by biofilm formation. By high-throughput phenotypic screening of small molecules, we identified compounds that inhibit adhesion of C. albicans to polystyrene. Our lead candidate compound also inhibits binding of C. albicans to cultured human epithelial cells, the yeast-to-hyphal morphological transition, induction of the hyphal-specific HWP1 promoter, biofilm formation on silicone elastomers, and pathogenesis in a nematode infection model as well as alters fungal morphology in a mouse mucosal infection assay. We term this compound filastatin based on its strong inhibition of filamentation, and we use chemical genetic experiments to show that it acts downstream of multiple signaling pathways. These studies show that high-throughput functional assays targeting fungal adhesion can provide chemical probes for study of multiple aspects of fungal pathogenesis.