Candida albicans (C. albicans) is a polymorphic fungus that can be a commensal part of the human microbiome. C. albicans can grow in the form of yeast, true hyphae, and pseudohyphae, the first two forms being pathogenic and thus causing an infection [1, 2]. C. albicans infections can be systemic, affecting the whole body or superficial, namely occurring as oral or vaginal candidiasis . This project focuses on vulvovaginal candidiasis (VVC), which is a fundamental worldwide public health problem . More than 80 % of VVC are caused solely by C. albicans . Acute infections are not dangerous but can be a nuisance, with the risk of developing a chronic infection.
Some women are more prone to VVC, namely those who experienced a previous colonization by the yeast, suffer from immunosuppressive diseases, diabetes mellitus or other factors like pregnancy or the use of antibiotics . All these factors promote either a decrease of other commensal organisms in the vaginal flora, or an excessive supply of nutrition. This contributes to an unbalanced ratio of microorganisms, in this case the overgrowth of C. albicans. It is estimated that worldwide around 75 % of women acquire the infection at least once, and 40-45 % experience two or more episodes [5, 6]. In Norway, there are no commercially available kits for detection of a C. albicans infection. Women suffering from an infection must either go to a physician for diagnosis or self-treat by purchasing antimycotics over-the-counter. Only 30 % of all women who self-treat with over-the-counter antimycotics in Norway have an actual Candida infection [A].
A study by Schwiertz et al. shows that the diagnosis of VVC by general practitioners and gynaecologists is poor. The misjudgement in this study is as high as 77,1 % [7, 8]. This unspecific treatment may lead to resistant microbes [A]. Antimicrobial resistance is a global problem and has dramatically increased over the past few years [World Health Organization]. This can lead to an uncontrollable spread and therefore a difficult treatment of infectious diseases [B]. With an easy to use test, antimicrobials could be provided specifically to patients who would profit from the treatment. Our product could contribute to the proper use of antimycotics and thus we could do our part in reducing antimicrobial resistance worldwide.
Taking advantage of the accurate CRISPR-Cas9 system, our team seeks to detect a specific DNA sequence of the C. albicans genome by using the dead Cas9 endonuclease (dCas9) protein. By inducing point mutations in the RuvC and HNH domain of the conventional Cas9, the endonuclease activity is inhibited. Designing guide RNAs that are complementary to 20 nucleotides of a gene, that is specific to C. albicans will allow the dCas9 to bind to the DNA strand that is being targeted. Since there is no endonuclease activity, the DNA strand is not getting cleaved [C].
By taking two dCas9 proteins and fusing each with one part of a split β-lactamase enzyme, a two-component system is created. Each component binds selectively to one target DNA sequence, which are both specific to the C. albicans genome. The near proximity of the two DNA target sequences allows the two parts of the β-lactamase, bound to the dCas9 to join and be enzymatically active again. Adding the substrate nitrocefin to the reaction solution that will be hydrolysed by the β-lactamase will provide a simple red colour readout suitable for the consumer [9, 10]. In order to obtain free C. albicans DNA we want to use an enzyme that selectively disrupts fungal cell walls, but not bacterial or human cells. Since the fungal cell wall mainly consists of polysaccharides, namely β-glucan and mannan sugar polymers. Glucanase is used to break down the glucan and thereby performing selective cell lysis . The sample will only contain free DNA of yeast, but no bacterial free DNA. This will eliminate a lot of false positive results.
Canditect in action
Animation by Nadeem Joudeh
Upon a suspected C. albicans infection, the woman goes to a physician or a pharmacist for diagnosis. The physician or pharmacist will use a sterile swab to collect a vaginal sample. This swab will then be inserted into the Canditect detection kit.
Inside the detection kit, glucanase enzymes catalyze the hydrolysis of the β-glucan bond in the yeast cell wall. Together with mechanical force, this will lyse the yeast cell wall, freeing the DNA. Two dCas9 enzymes, each fused to a split β-lactamase, will recognize their target sequence in the C. albicans DNA. When the dCas9 binds the DNA, the β-lactamase will be functional again, converting its substrate, nitrocefin, to a hydrolyzed product with a red color. This will yield an easy color readout, which can be observed with the naked eye in the detection kit window.
The beauty of this system is its sheer flexibility. By changing the guide RNA, we can adapt the system to detect almost any organism containing DNA, with high accuracy. If we are successful, our system can be readily applied to cover diagnostics for several diseases.
- A: http://www.antibiotikaiallmennpraksis.no/index.php?action=showtopic&topic=DrmsfZGV accessed at 22.08.2018
- B: http://www.who.int/antimicrobial-resistance/en/ , accessed at 19.08.2018
- C: https://www.addgene.org/crispr/guide/ , accessed at 20.08.2018