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A synthetic system that senses Candida albicans and inhibits virulence factors Michael Tscherner, Tobias Wolfgang Giessen, Laura Markey, Carol Kumamoto, and Pamela A Silver ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00457 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019
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ACS Synthetic Biology
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A synthetic system that senses Candida albicans and inhibits virulence
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factors
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Michael Tscherner1,2,§, Tobias W. Giessen1,2, Laura Markey3, Carol A. Kumamoto3 and
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Pamela A. Silver1,2*
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1Department
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2Wyss
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Massachusetts, USA
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3Graduate
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Sciences and Department of Molecular Biology and Microbiology, Tufts University, Boston,
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Massachusetts, USA
of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA
Institute
for
Biologically
Inspired
Engineering,
Harvard
University,
Boston,
Program in Molecular Microbiology, Sackler School of Graduate Biomedical
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§current
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Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria
address: Medical University of Vienna, Center for Medical Biochemistry, Max F.
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Abstract
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Due to a limited set of antifungals available and problems in early diagnosis invasive fungal
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infections caused by Candida species are among the most common hospital-acquired
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infections with staggering mortality rates. Here, we describe an engineered system able to
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sense and respond to the fungal pathogen Candida albicans, the most common cause of
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candidemia. In doing so, we identified hydroxyphenylacetic acid (HPA) as a novel molecule
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secreted by C. albicans. Furthermore, we engineered E. coli to be able to sense HPA
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produced by C. albicans. Finally, we constructed a sense-and-respond system by coupling
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the C. albicans sensor to the production of an inhibitor of hypha formation thereby reducing
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filamentation, virulence factor expression and fungal-induced epithelial damage. This system
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could be used as a basis for the development of novel prophylactic approaches to prevent
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fungal infections.
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Keywords: Candida albicans, hypha inhibition, engineered probiotics, sense-and-respond
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Fungal pathogens cause diverse types of infections ranging from superficial to systemic and
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claim about 1.5 million lives per year1. Candida species are among the most common
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opportunistic fungal pathogens and represent the fourth-leading cause of hospital-acquired
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bloodstream infections with mortality rates of more than 40% in immunocompromised
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patients1–4.
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Candida albicans represents the main cause of candidemia, being responsible for
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more than 40% of all cases worldwide2. C. albicans is a commensal colonizing the
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gastrointestinal
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immunocompromised conditions, C. albicans can penetrate epithelia and cause severe
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systemic infections6. A key virulence factor of C. albicans is its morphologic plasticity; it can
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reversibly switch between a yeast and a filamentous or true hyphal morphology depending on
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the environmental conditions. On epithelial surfaces, initial adhesion and subsequent
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filamentation are required for efficient epithelial penetration7. In addition, several virulence
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factors such as Candidalysin, a peptide that damages epithelial cells, are produced
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exclusively by the hyphal form8.
and
genitourinary
tracts
of
healthy
individuals5.
However,
under
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Difficulties associated with rapid and reliable diagnosis as well as lack of specific
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disease manifestations contribute to the high mortality rates of systemic fungal infections9.
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Antifungal prophylaxis is currently applied to high risk individuals. While effective in preventing ACS Paragon Plus Environment
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Candida infections10,11, prolonged prophylaxis can lead to the emergence of resistant strains
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or to an increase of species, that are intrinsically resistant to the limited set of antifungals
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available10,12. Development of novel antifungals with fungistatic or fungicidal activity is limited
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by the relative dearth of specific targets in these eukaryotic pathogens. Thus, more recently
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approaches targeting specific virulence factors have been gaining interest. This strategy can
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increase the number of potential targets, that are specific to the pathogen, and will exert less
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evolutionary pressure for the development of resistance compared to growth inhibition. In the
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case of C. albicans, inhibition of filamentation represents an important target because it could
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prevent physical penetration of epithelia as well as expression of hypha-specific virulence
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genes7.
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A number of studies have shown that members of the human microbiota can inhibit
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hypha formation by C. albicans13–16. However, the inhibitory substance is usually produced
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constitutively and does not represent a specific response to the presence of the fungus. One
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example of a bacterial secreted molecule that efficiently blocks hypha formation by C.
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albicans, is cis-2-dodecenoic acid (BDSF), a diffusible signal factor that is produced by
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Burkholderia cenocepacia13.
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Synthetic
biology
approaches
using
genetically
engineered
bacteria
have
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demonstrated great potential in the prophylaxis or treatment of cancer, metabolic disorders
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and infectious diseases17–20. Using engineered microbes offers several advantages over
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traditional therapeutics including lower cost and higher specificity due to more targeted
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treatment17. A number of approaches using engineered bacteria to counteract or prevent
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bacterial and viral infections have been reported21–26. However, there have been no reports
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describing the use of reprogrammed microorganisms for treatment of or prophylaxis against
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fungal infections.
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In this study, we developed an engineered system able to sense a fungal pathogen
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and respond by inhibition of fungal virulence factors. We discovered hydroxyphenylacetic acid
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(HPA) as a novel molecule secreted by C. albicans and used engineered E. coli cells carrying
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a HPA sensor to detect the presence of the fungus. Furthermore, we developed a sense-and-
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respond system by coupling the sensor to the production of BDSF to inhibit filamentation and
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virulence gene expression in C. albicans thereby protecting epithelial cells from fungal-
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mediated damage.
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Results
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System design
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The overall goal of this work was to construct and characterize an engineered, commensal E.
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coli strain capable of first, detecting the presence of C. albicans and second, producing a
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hypha inhibitor in response to detection thereby preventing filamentation (Figure 1). The
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sensor should be able to sense a substance secreted by the fungus and control expression of
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a hypha inhibition module, which will lead to the production of an inhibitor of hypha formation.
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Thus, bacteria carrying this system would be able to protect an epithelium from being
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damaged by C. albicans.
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Figure 1. Design of a C. albicans sensing and hypha inhibition system. C. albicans can penetrate
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host epithelia by switching from a yeast to a hyphal morphology. Engineered E. coli cells can sense C.
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albicans secreted molecules and respond by production of an inhibitor of hypha formation thereby
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preventing epithelial damage and penetration.
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Engineered E. coli can sense a molecule produced by C. albicans
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To engineer E. coli to sense C. albicans, we first focused our efforts on the known fungal
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quorum sensing molecule tyrosol (4-hydroxyphenylethanol), which is produced in different
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morphologies and even at sites of infection27. E. coli W strains are able to launch a
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transcriptional response to the structurally related substance 4-hydroxyphenylacetic acid (4-
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HPA)28. Therefore, we reasoned that the structural similarity between tyrosol and 4-HPA
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could serve as a starting point for the construction of a tyrosol sensor.
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We first tested for tyrosol production by C. albicans. We harvested C. albicans culture
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supernatants and confirmed tyrosol production by HPLC-MS, which increased upon
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supplementation of the medium with its precursor tyrosine (Figure S1a). As a control we also
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determined the levels of HPA in fungal culture supernatants. Surprisingly, we detected HPA
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production by C. albicans and the levels increased upon tyrosine supplementation (Figure
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2a). Furthermore, we could detect HPA in yeast and hyphal supernatants (Figure S1b).
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Based on its retention time we were able to identify the secreted molecule as being either 3-
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HPA or 4-HPA. Thus, C. albicans is able to produce HPA presumably via a pathway similar to
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tyrosol.
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E. coli has the ability to respond to 4-HPA and can be engineered to record its
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production by C. albicans. The E. coli 4-HPA sensor found in W strains consists of a
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transporter (HpaX) and a transcription factor (HpaA)28,29. Upon import by HpaX and binding to
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HpaA, 4-HPA can activate transcription from the downstream PBC promoter28.To test this
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system, we constructed a sensor plasmid carrying the hpaX and hpaA genes and the PBC
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promoter upstream of a gfp encoding gene (Figure 2b). Cells carrying the sensor plasmid
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(PAS691) showed increased GFP expression upon induction with 4-HPA (Figure S1c).
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Furthermore, the absence of the HpaX transporter (PAS692) decreased the sensitivity of the
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sensor. Tyrosol did not lead to efficient induction even in the presence of HpaX. To determine
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if the 4-HPA sensor can detect C. albicans, we incubated E. coli PAS691 with different
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dilutions of fungal supernatants and measured GFP levels after 24 hours (Figure 2b). C.
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albicans culture supernatants were able to strongly induce the 4-HPA sensor and tyrosine
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supplementation of the fungal culture further increased the induction level.
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Figure 2. Construction of a C. albicans sensor. (a) C. albicans produces hydroxyphenylacetic acid
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(HPA). HPA levels in culture supernatants were determined by HPLC-MS. Addition of tyrosine
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increases HPA production. Means of biological replicates are indicated. One-tailed Student's t-test:
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**P
