Project Description

hyphae

Background

Candida is a genus of yeast that is the most common cause of fungal infections globally1. One of the more lethal species is Candida auris, which has an estimated mortality rate of 30-40%2. While it is dangerous, it is not the most common reason for fungal infections. That title belongs to Candida albicans3.

Candida albicans is an opportunistic fungal species that naturally resides on human skin and mucosal surfaces. It is polymorphic, meaning that it can exist as both individual cells and in a hyphal form, closer to how filamentous fungi grow. Under certain conditions—such as changes in the host immune response during stress, co-infection with another microbe, antibiotic treatment, or immunosuppressive therapy—C. albicans can overgrow and transition from harmless to life-threatening4. When any species of Candida causes an infection, the disease is commonly referred to as candidiasis5. C. albicans accounts for 65% of fungal infections worldwide3. The most commonly encountered infections are superficial; while not deadly, they affect a large number of people and create a significant burden on healthcare systems globally6. For example, recurrent vulvovaginal candidiasis (RVVC) alone causes approximately 140 million cases per year, affecting an estimated 8% of women annually worldwide, and up to 75% of women experience vulvovaginal candidiasis at least once in their lifetime7. In more severe cases, candidiasis can be fatal6.

Current antifungal treatments are limited by systemic toxicity, long treatment courses, poor selectivity, rising resistance, and disruption of the natural microbiome—highlighting an urgent need for targeted, sustainable, and patient-friendly antifungal therapies. The main challenge of antifungal drugs lies in the similarities between fungal cells and human cells, which can cause the therapies to inadvertently target our own bodies4.

Our Project

In support of the third United Nations Sustainable Development Goal—to “Ensure healthy lives and promote well-being for all at all ages”8—our project aims to develop a new treatment for candidiasis. This treatment would leverage the body's own defense system against fungal infections and enhance it, replacing some of the current treatments that cause unwanted side effects.

The treatment is based on Bacillus subtilis, a non-pathogenic bacterium that lives on human skin. It has been shown to produce an antifungal substance called Iturin A, which can combat Candida9. In its wild-type state, B. subtilis does not produce enough Iturin A to treat a larger infection effectively. However, if modified to attach to fungal cells, it could increase the local concentration of Iturin A. Efforts are already underway to increase Iturin A production in B. subtilis10, but delivery methods remain underexplored.

Previously, Escherichia coli was engineered to attach to chitin in the cell wall of C. albicans using specific proteins. A modular construct was created that includes an anchor protein attached to the bacterial membrane, a linker chain, and a terminal binding protein11.

The terminal protein in this linker system can be substituted, giving us a bacterial display system. The Candida cell wall is rich in chitin, which contributes to its pathogenicity. Chitin binds to a class of proteins called chitinases. Engineering B. subtilis to express chitinases on its membrane could allow it to bind to Candida and deliver Iturin A directly to the pathogen.

A schematic of this display system is shown below.

Simplified schematic of the chitinase display system.

Wet lab

To enable B. subtilis to produce chitinases, we designed a plasmid containing the display system described in Chamas et al.11, incorporating the chitinase gene CHI92. The same study also mentioned a synthetic protein called SP1, which binds to chitin. We designed a second plasmid with SP1 as the binding protein to test alongside CHI92. More information about the specific design can be found on the PARTS page.

To enable B. subtilis to produce chitinases, we designed a plasmid containing the display system described in11, incorporating the chitinase gene CHI92. The same study also mentioned a synthetic protein called SP1, which binds to chitin. We designed a second plasmid with SP1 as the binding protein to test alongside CHI92. More information about the specific design can be found on the PARTS page.

Verification of these constructs cannot be performed on C. albicans because it requires a BSL-2 lab12, which we do not have access to. To address this, we can create a model organism. Like C. albicans, baker's yeast (Saccharomyces cerevisiae) contains chitin in its cell wall, although in smaller amounts. By introducing the native S. cerevisiae chitin synthesis gene, CHS3, into a plasmid, we can increase the chitin content in the membrane since the necessary pathways already exist in the cells. A third plasmid was therefore designed, containing the CHS3 gene.

The binding affinity of B. subtilis to chitin can be tested in several ways. One approach uses a column filled with chitin-coated beads. A liquid culture of modified B. subtilis is added to the top of the column and allowed to interact with the beads before being collected at the bottom. By counting the cells in the liquid before and after using a hemocytometer, binding efficiency can be calculated. Wild-type bacteria serve as the control group for comparison.

Measuring binding affinity using chitin-coated beads.


To test binding to live cells, a co-culture of B. subtilis and S. cerevisiae can be created. Using microscopy, we can estimate how many cells appear to adhere to one another. Comparing these results to co-cultures of modified and wild-type cells allows for statistical analysis.

Modified B. subtilis and S. cerevisiae.
Wild-type B. subtilis and modified S. cerevisiae.
Modified B. subtilis and wild-type S. cerevisiae.

Although measuring fungal cell death is beyond the scope of this project, it would be highly interesting to determine whether B. subtilis kills more C. albicans when it can adhere to them. Combining this with increased Iturin A production in the bacteria could form the basis for further project development.

Dry lab

Besides experimental approaches, we conducted computational modeling to investigate the binding behavior of chitinase-92 and SP1 with small chitin fragments, including chitobiose, chitotriose, and N-acetyl-β-D-glucosamine dimers and trimers. To achieve this, we first predicted the 3D structures of the binding sites using AlphaFold2 as implemented in ColabFold13, 14. These models were then used in AutoDock 4.2.615 to estimate binding energies and identify potential interaction sites. This workflow provided a quantitative assessment of SP1's binding capability and a theoretical estimate of how effectively the engineered bacteria could adhere to chitin surfaces, and provide a confirmatory analysis of the qualitative evaluation of SP1's binding characteristics, that carbohydrate binding of SP1 to chitin partially relies on hydrophobic interactions involving the peptide's aromatic amino acids16. Furthermore, the topology of SP1, with several β-sheets arranged in a planar architecture, is characteristic of type A carbohydrate-binding modules that bind crystalline chitin11, providing a structural explanation for SP1's strong binding properties.

Inspiration

The primary inspiration for our project came from firsthand experience with antifungal therapies for onychomycosis by one of our team members. This experience provided insight into the limitations of current antifungal treatments. When C. albicans and Iturin A came up during our initial research, the combination felt like a relevant and promising fit for us—especially considering that fungal infections are often overlooked compared to other equally harmful diseases17.

References

  1. Brandt, M. E., Lockhart, S. R., Recent Taxonomic Developments in the Genus Candida and Other Opportunistic Yeast. Current fungal infection reports. 2012;6(3):170–177.
  2. Eix, E. F., & Nett, J. E., Candida auris: Epidemiology and Antifungal Strategy. Annual review of medicine. 2025;76(1):57–67.
  3. Morad HOJ, Wild AM, Wiehr S, Davies G, Maurer A, Pichler BJ, et al. Pre-clinical Imaging of Invasive Candidiasis Using ImmunoPET/MR. Front Microbiol. 2018;9:1996.
  4. Nobile CJ, Johnson AD. Candida albicans Biofilms and Human Disease. Annu Rev Microbiol. 2015;69:71-92.
  5. CDC. Candidiasis. 2024 [cited Oct 3 2025]. Candidiasis Basics. Available from: https://www.cdc.gov/candidiasis/about/index.html
  6. Talapko J, Juzbašić M, Matijević T, Pustijanac E, Bekić S, Kotris I, et al. Candida albicans-The Virulence Factors and Clinical Manifestations of Infection. J Fungi (Basel). 2021 Jan 22;7(2):79.
  7. Willems HME, Ahmed SS, Liu J, Xu Z, Peters BM. Vulvovaginal Candidiasis: A Current Understanding and Burning Questions. J Fungi (Basel). 2020 Feb 25;6(1):27.
  8. Goal 3 | Department of Economic and Social Affairs [Internet]. [cited 2025 Oct 3]. Available from: https://sdgs.un.org/goals/goal3
  9. Lei S, Zhao H, Pang B, Qu R, Lian Z, Jiang C, et al. Capability of iturin from Bacillus subtilis to inhibit Candida albicans in vitro and in vivo. Appl Microbiol Biotechnol. 2019 Jun 1;103(11):4377-92.
  10. Yue H, Zhong J, Li Z, Zhou J, Yang J, Wei H, et al. Optimization of iturin A production from Bacillus subtilis ZK-H2 in submerged fermentation by response surface methodology. 3 Biotech. 2021 Feb;11(2):36.
  11. Chamas A, Svensson CM, Maneira C, Sporniak M, Figge MT, Lackner G. Engineering Adhesion of the Probiotic Strain Escherichia coli Nissle to the Fungal Pathogen Candida albicans. ACS Synth Biol. 2024 Dec 20;13(12):4027-39.
  12. Biosafety Levels for Biological Agents - Stanford Environmental Health & Safety [Internet]. [cited 2025 Oct 3]. Available from: https://ehs.stanford.edu/reference/biosafety-levels-biological-agents
  13. Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583-589.
  14. Mirdita M, Schütze K, Moriwaki Y, et al. ColabFold: making protein folding accessible to all. Nature Methods. 2022;19:679-682.
  15. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. J Comput Chem. 2009;30(16):2785-2791.
  16. Hudson KL, Bartlett GJ, Diehl RC, Agirre J, Gallagher T, Kiessling LL, Woolfson DN. Carbohydrate-aromatic interactions in proteins. J Am Chem Soc. 2015;137:15152-15160.
  17. Rodrigues ML, Nosanchuk JD. Fungal diseases as neglected pathogens: A wake-up call to public health officials. PLoS Negl Trop Dis. 2020 Feb 20;14(2).