A Synthetic System That Senses Candida albicans ... - ACS Publications

(5) However, under immunocompromised conditions, C. albicans can penetrate epithelia and cause severe systemic infections.(6) A key virulence factor o...
0 downloads 0 Views 944KB Size
Subscriber access provided by University of Kansas Libraries

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1 2 3 4 5

A synthetic system that senses Candida albicans and inhibits virulence

6

factors

7 8

Michael Tscherner1,2,§, Tobias W. Giessen1,2, Laura Markey3, Carol A. Kumamoto3 and

9

Pamela A. Silver1,2*

10 11

1Department

12

2Wyss

13

Massachusetts, USA

14

3Graduate

15

Sciences and Department of Molecular Biology and Microbiology, Tufts University, Boston,

16

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

17 18

§current

19

Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria

address: Medical University of Vienna, Center for Medical Biochemistry, Max F.

20

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 27

21

Abstract

22

Due to a limited set of antifungals available and problems in early diagnosis invasive fungal

23

infections caused by Candida species are among the most common hospital-acquired

24

infections with staggering mortality rates. Here, we describe an engineered system able to

25

sense and respond to the fungal pathogen Candida albicans, the most common cause of

26

candidemia. In doing so, we identified hydroxyphenylacetic acid (HPA) as a novel molecule

27

secreted by C. albicans. Furthermore, we engineered E. coli to be able to sense HPA

28

produced by C. albicans. Finally, we constructed a sense-and-respond system by coupling

29

the C. albicans sensor to the production of an inhibitor of hypha formation thereby reducing

30

filamentation, virulence factor expression and fungal-induced epithelial damage. This system

31

could be used as a basis for the development of novel prophylactic approaches to prevent

32

fungal infections.

33 34

Keywords: Candida albicans, hypha inhibition, engineered probiotics, sense-and-respond

35 36

Fungal pathogens cause diverse types of infections ranging from superficial to systemic and

37

claim about 1.5 million lives per year1. Candida species are among the most common

38

opportunistic fungal pathogens and represent the fourth-leading cause of hospital-acquired

39

bloodstream infections with mortality rates of more than 40% in immunocompromised

40

patients1–4.

41

Candida albicans represents the main cause of candidemia, being responsible for

42

more than 40% of all cases worldwide2. C. albicans is a commensal colonizing the

43

gastrointestinal

44

immunocompromised conditions, C. albicans can penetrate epithelia and cause severe

45

systemic infections6. A key virulence factor of C. albicans is its morphologic plasticity; it can

46

reversibly switch between a yeast and a filamentous or true hyphal morphology depending on

47

the environmental conditions. On epithelial surfaces, initial adhesion and subsequent

48

filamentation are required for efficient epithelial penetration7. In addition, several virulence

49

factors such as Candidalysin, a peptide that damages epithelial cells, are produced

50

exclusively by the hyphal form8.

and

genitourinary

tracts

of

healthy

individuals5.

However,

under

51

Difficulties associated with rapid and reliable diagnosis as well as lack of specific

52

disease manifestations contribute to the high mortality rates of systemic fungal infections9.

53

Antifungal prophylaxis is currently applied to high risk individuals. While effective in preventing ACS Paragon Plus Environment

Page 3 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

54

Candida infections10,11, prolonged prophylaxis can lead to the emergence of resistant strains

55

or to an increase of species, that are intrinsically resistant to the limited set of antifungals

56

available10,12. Development of novel antifungals with fungistatic or fungicidal activity is limited

57

by the relative dearth of specific targets in these eukaryotic pathogens. Thus, more recently

58

approaches targeting specific virulence factors have been gaining interest. This strategy can

59

increase the number of potential targets, that are specific to the pathogen, and will exert less

60

evolutionary pressure for the development of resistance compared to growth inhibition. In the

61

case of C. albicans, inhibition of filamentation represents an important target because it could

62

prevent physical penetration of epithelia as well as expression of hypha-specific virulence

63

genes7.

64

A number of studies have shown that members of the human microbiota can inhibit

65

hypha formation by C. albicans13–16. However, the inhibitory substance is usually produced

66

constitutively and does not represent a specific response to the presence of the fungus. One

67

example of a bacterial secreted molecule that efficiently blocks hypha formation by C.

68

albicans, is cis-2-dodecenoic acid (BDSF), a diffusible signal factor that is produced by

69

Burkholderia cenocepacia13.

70

Synthetic

biology

approaches

using

genetically

engineered

bacteria

have

71

demonstrated great potential in the prophylaxis or treatment of cancer, metabolic disorders

72

and infectious diseases17–20. Using engineered microbes offers several advantages over

73

traditional therapeutics including lower cost and higher specificity due to more targeted

74

treatment17. A number of approaches using engineered bacteria to counteract or prevent

75

bacterial and viral infections have been reported21–26. However, there have been no reports

76

describing the use of reprogrammed microorganisms for treatment of or prophylaxis against

77

fungal infections.

78

In this study, we developed an engineered system able to sense a fungal pathogen

79

and respond by inhibition of fungal virulence factors. We discovered hydroxyphenylacetic acid

80

(HPA) as a novel molecule secreted by C. albicans and used engineered E. coli cells carrying

81

a HPA sensor to detect the presence of the fungus. Furthermore, we developed a sense-and-

82

respond system by coupling the sensor to the production of BDSF to inhibit filamentation and

83

virulence gene expression in C. albicans thereby protecting epithelial cells from fungal-

84

mediated damage.

85 86 ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 27

87

Results

88

System design

89

The overall goal of this work was to construct and characterize an engineered, commensal E.

90

coli strain capable of first, detecting the presence of C. albicans and second, producing a

91

hypha inhibitor in response to detection thereby preventing filamentation (Figure 1). The

92

sensor should be able to sense a substance secreted by the fungus and control expression of

93

a hypha inhibition module, which will lead to the production of an inhibitor of hypha formation.

94

Thus, bacteria carrying this system would be able to protect an epithelium from being

95

damaged by C. albicans.

96

97 98

Figure 1. Design of a C. albicans sensing and hypha inhibition system. C. albicans can penetrate

99

host epithelia by switching from a yeast to a hyphal morphology. Engineered E. coli cells can sense C.

100

albicans secreted molecules and respond by production of an inhibitor of hypha formation thereby

101

preventing epithelial damage and penetration.

102 103

ACS Paragon Plus Environment

Page 5 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

104 105

Engineered E. coli can sense a molecule produced by C. albicans

106

To engineer E. coli to sense C. albicans, we first focused our efforts on the known fungal

107

quorum sensing molecule tyrosol (4-hydroxyphenylethanol), which is produced in different

108

morphologies and even at sites of infection27. E. coli W strains are able to launch a

109

transcriptional response to the structurally related substance 4-hydroxyphenylacetic acid (4-

110

HPA)28. Therefore, we reasoned that the structural similarity between tyrosol and 4-HPA

111

could serve as a starting point for the construction of a tyrosol sensor.

112

We first tested for tyrosol production by C. albicans. We harvested C. albicans culture

113

supernatants and confirmed tyrosol production by HPLC-MS, which increased upon

114

supplementation of the medium with its precursor tyrosine (Figure S1a). As a control we also

115

determined the levels of HPA in fungal culture supernatants. Surprisingly, we detected HPA

116

production by C. albicans and the levels increased upon tyrosine supplementation (Figure

117

2a). Furthermore, we could detect HPA in yeast and hyphal supernatants (Figure S1b).

118

Based on its retention time we were able to identify the secreted molecule as being either 3-

119

HPA or 4-HPA. Thus, C. albicans is able to produce HPA presumably via a pathway similar to

120

tyrosol.

121

E. coli has the ability to respond to 4-HPA and can be engineered to record its

122

production by C. albicans. The E. coli 4-HPA sensor found in W strains consists of a

123

transporter (HpaX) and a transcription factor (HpaA)28,29. Upon import by HpaX and binding to

124

HpaA, 4-HPA can activate transcription from the downstream PBC promoter28.To test this

125

system, we constructed a sensor plasmid carrying the hpaX and hpaA genes and the PBC

126

promoter upstream of a gfp encoding gene (Figure 2b). Cells carrying the sensor plasmid

127

(PAS691) showed increased GFP expression upon induction with 4-HPA (Figure S1c).

128

Furthermore, the absence of the HpaX transporter (PAS692) decreased the sensitivity of the

129

sensor. Tyrosol did not lead to efficient induction even in the presence of HpaX. To determine

130

if the 4-HPA sensor can detect C. albicans, we incubated E. coli PAS691 with different

131

dilutions of fungal supernatants and measured GFP levels after 24 hours (Figure 2b). C.

132

albicans culture supernatants were able to strongly induce the 4-HPA sensor and tyrosine

133

supplementation of the fungal culture further increased the induction level.

134

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 27

135 136

Figure 2. Construction of a C. albicans sensor. (a) C. albicans produces hydroxyphenylacetic acid

137

(HPA). HPA levels in culture supernatants were determined by HPLC-MS. Addition of tyrosine

138

increases HPA production. Means of biological replicates are indicated. One-tailed Student's t-test:

139

**P