nihms626355.pdf

Anaerobic Bacteria Grow within

Candida albicans

Biofilms and

Induce Biofilm Formation in Suspension Cultures

Emily P. Fox

1,2

Elise S. Cowley

3,4

Clarissa J. Nobile

1,5

Nairi Hartooni

Dianne K.

Newman

3,4

, and

Alexander D. Johnson

Department of Microbiology and Immunology, University of California, San Francisco, 600 16

Street, San Francisco, CA, United States of America

Tetrad Program, Department of Biochemistry and Biophysics, University of California, San

Francisco, 600 16

Street, San Francisco, CA, United States of America

Division of Biology and Biological Engineering, California Institute of Technology, 147-75, 1200E

California Boulevard, Pasadena, CA, United States of America

Howard Hughes Medical Institute, California Institute of Technology, 147-75, 1200E California

Boulevard, Pasadena, CA, United States of America

School of Natural Sciences, University of California, Merced, 5200 North Lake Road, Merced,

CA, United States of America

Summary

The human microbiome contains diverse microorganisms, which share and compete for the same

environmental niches [

]. A major microbial growth form in the human body is the biofilm

state, where tightly packed bacterial, archaeal and fungal cells must cooperate and/or compete for

resources in order to survive [

–

]. We examined mixed biofilms composed of the major fungal

species of the gut microbiome,

C. albicans,

and each of five prevalent bacterial gastrointestinal

inhabitants:

Bacteroides fragilis

Clostridium perfringens

Escherichia coli, Klebsiella

pneumoniae

and

Enterococcus faecalis

[

–

]. We observed that biofilms formed by

C. albicans

provide a hypoxic microenvironment that supports the growth of two anaerobic bacteria, even

when cultured in ambient oxic conditions that are normally toxic to the bacteria. We also found

that co-culture with bacteria in biofilms induces massive gene expression changes in

C. albicans

including upregulation of

WOR1,

which encodes a transcription regulator that controls a

phenotypic switch in

C. albicans,

from the “white” cell type to the “opaque” cell type. Finally, we

observed that in suspension cultures,

C. perfringens

induces aggregation of

C. albicans

into “mini-

biofilms,” which allow

C. perfringens

cells to survive in a normally toxic environment. This work

indicates that bacteria and

C. albicans

interactions modulate the local chemistry of their

environment in multiple ways to create niches favorable to their growth and survival.

Address correspondence to: Alexander D. Johnson, ajohnson@cgl.ucsf.edu.

Publisher's Disclaimer:

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our

customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of

the resulting proof before it is published in its final citable form. Please note that during the production process errors may be

discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public Access

Author Manuscript

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

Published in final edited form as:

Curr Biol

. 2014 October 20; 24(20): 2411–2416. doi:10.1016/j.cub.2014.08.057.

NIH-PA Author Manuscript

Results

The fungal species

C. albicans

forms mixed biofilms with five bacterial species

C. albicans

with or without

C. perfringens, B. fragilis

E. faecalis, E. coli

K. pneumoniae

cells were adhered to a bovine serum coated, polystyrene well for 90 minutes and allowed to

develop into biofilms for 24 hours, a standard procedure for producing

C. albicans

biofilms

[

]. Confocal scanning laser microscopy (CSLM) images confirmed that in all cases,

both fungal and bacterial species incorporated into the biofilm (Figure 1). The bacteria

adhered to both

C. albicans

hyphal and yeast-form cells (Figure 1; Figure S1A – F). While

B. fragilis,

and

C. perfringens

had minimal effect on the biofilm architecture, incorporation

E. coli, E. faecalis

and

K. pneumoniae

reduced the overall biofilm thickness (Figure

S1G). We designed a colony forming unit (CFU) assay as a readout for live bacterial and

albicans

cells present, and found that both bacteria and

C. albicans

were incorporated into

the biofilms over time (Figure 2A – D, S2A – C).

C. perfringens

and

B. fragilis

proliferate in co-cultured biofilms with

C. albicans

under

ambient oxic conditions

C. albicans

and/or

C. perfringens

B. fragilis

cells were co-cultured in biofilms for 4, 24,

48, or 72 h, under ambient oxic or anoxic conditions. Growth of each species over time was

measured by plating for CFUs (Figure 2A – D). The adherence and growth of

C. albicans

was unaffected by the presence or absence of bacterial cells; however the initial adherence

C. perfringens

and

B. fragilis

increased ten-fold in the presence of

C. albicans.

In mixed

biofilms

after adherence,

C. perfringens

showed substantial growth, from ~5×10

CFU/ml

to ~1×10

CFU/ml in 24 h, regardless of whether the biofilm was grown under ambient oxic

or anoxic conditions (Figure 2A, C). Without

C. albicans

, viable

C. perfringens

cells

decreased below detection (<10 CFU/ml) after 24 h in ambient oxic conditions (Figure 2A).

B. fragilis

showed the same trend (Figure 2B, D). In addition to the standard laboratory

strain of

C. albicans

(SC5314), we tested two other clinical isolates of

C. albicans

and found

they are also able to support anaerobe growth (Figure S2D, E). Our data demonstrate that

incorporation into a

C. albicans

biofilm grown under ambient oxic conditions enables

growth of the anaerobes

C. perfringens

and

B. fragilis

; without the protective biofilm, the

viability of both bacterial species rapidly declines.

C. albicans

biofilms create a hypoxic microenvironment

To test the hypothesis that biofilms create locally hypoxic environments which enable the

growth of anaerobic bacteria, we measured oxygen levels in biofilms using a miniaturized,

Switch-able Trace Oxygen Sensor (STOX-Sensor), an instrument capable of measuring

oxygen concentrations as low as 10 nM [

]. Measurements with the STOX-Sensor revealed

a gradient of oxygen concentration throughout the depth of the biofilm, decreasing from

~300 μM (ambient oxygen) near the top of the biofilm to less than 50 μM near the bottom

(Figure 2E). The oxygen gradient remained the same whether

C. albicans

was grown in

monoculture or was co-cultured with

C. perfringens

B. fragilis.

Fox et al.

Page 2

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

Co-culture in biofilms with bacteria alters gene expression in

C. albicans

To determine whether

C. albicans

was responding to bacteria in the mixed-species biofilm,

we measured gene expression changes in

C. albicans

by microarray (Figure 3A; Dataset 1).

Relative to the

C. albicans

biofilm formed in the absence of bacteria, many genes were up-

and down-regulated in the presence of bacteria. Some genes changed expression in response

to all of the bacterial species, while others were specific to a few species.

Among the most differentially regulated genes were those encoding the transcription

regulators controlling the white-opaque switch in

C. albicans

, a transition between two cell

types, each of which is heritable for many generations [

–

] (Figure 3B). In particular,

WOR1

, which encodes the “master” regulator of white-opaque switching, was strongly

upregulated by co-culture with

K. pneumoniae, E. coli,

and

E. faecalis.

Co-culture with

pneumoniae

also induced upregulation of several other transcription regulators known to

play roles in the white-opaque switch, in a

WOR1

-independent manner (Figure S3, Dataset

2) [

–

Although a number of opaque-specific genes were upregulated, the full opaque-specific

gene expression pattern was not observed, and when removed from this condition, the

albicans

cells revert to “classical” white cells. We propose that co-culture with bacterial

cells poises

C. albicans

to switch from white to opaque, but that additional signals are

required for full switching.

C. perfringens

is protected by and induces aggregation of

C. albicans

in suspension

culture

To further explore interactions between

C. albicans

and the bacterial microbiome members,

we co-cultured them in suspension cultures, and observed that some of the bacteria induced

co-aggregation with

C. albicans

cells (Table S1, Figure 4A – D). The most dramatic effect

occurred with

C. perfringens

in ambient oxic conditions. Light microscopy revealed that the

aggregates induced by

C. perfringens

were composed of dense clumps containing both

albicans

and

C. perfringens

cells and resembling miniature biofilms (Figure 4G). By

monitoring CFUs/ml of

C. perfringens

grown in suspension cultures over time (Figure 4H,

I), we observed that the presence of

C. albicans

enabled survival of

C. perfringens

in oxic

suspension conditions to levels of ~1×10

CFU/ml; in the absence of

C. albicans, C.

perfringens

CFUs dropped at least five orders of magnitude, to undetectable levels (<10

CFU/ml) by 24 h (Figure 4H).

Although the mini-biofilms are too small to directly probe for oxygen concentration, we

note that

C. albicans

gene expression under these conditions was significantly enriched for

genes regulated during hypoxic conditions (P = 1.4×10

−5

) [

] (Figure S4A, Dataset 3),

suggesting that the mini-biofilms, like conventional, surface-adhered biofilms, provide a

hypoxic environment. Consistent with this idea, we found that

C. perfringens

cells also

stimulate aggregation in early stages of conventional

C. albicans

biofilm formation on a

solid surface (Figure S4B).

We repeated the suspension growth experiment with cell-free supernatant or heat-killed

perfringens

cells, and observed that both are able to induce aggregation of

C. albicans

Fox et al.

Page 3

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

(Figure 4E, F). We blindly screened a library of 205 deletion strains in

C. albicans

[

]

(Table S2), and identified eight transcription regulator-encoding genes and two other genes

that are required for the observed interspecies aggregation (Figure 4K–R; Figure S4C).

Notably, six of the transcription regulators (Brg1, Tec1, Rob1, Bcr1, Ndt80, and Efg1)

found in our screen were previously identified “master regulators” of conventional biofilm

formation [

], providing strong evidence that

C. perfringens

induces aggregate formation

via the biofilm genetic program. The other two regulator mutants deficient in aggregation

were

rim101

Δ/Δ and

flo8

Δ/Δ, which have not been reported to be required for conventional

biofilm formation.

DEF1

, which regulates hyphal extension [

], and

ALS3,

which encodes

an adhesin important for biofilm formation and plays a role in interacting with many

bacterial species [

–

], were also required for aggregation (Figure S4C). As described in

supplemental materials, we quantified aggregation using a sedimentation assay and verified

that the deletion strains were complemented by gene “add-backs” (Figure S4D, E).

These results support a model whereby in ambient oxic suspension culture,

C. perfringens

induces

C. albicans

to form protective aggregates, which depend on the

C. albicans

biofilm

genetic program. These mini-biofilms, which contain both

C. albicans

and

C. perfringens

allow

C. perfringens

to survive in oxic conditions that are normally toxic.

Discussion

In this work we uncovered multiple interactions between

C. albicans,

a major fungal species

of the human microbiome, and several bacterial members of the microbiome.

C. albicans

biofilms: a microenvironment supporting anaerobic bacterial growth

It has been known for some time that bacterial biofilms are able to generate hypoxic

microenvironments, supporting the growth of anaerobic bacterial species [

], and it has

been speculated that biofilms formed by

Candida

species may also be hypoxic, based on

gene expression data and mutant phenotypes [

–

]. Our work directly demonstrates,

for the first time, that

C. albicans

biofilms create a hypoxic internal microenvironment when

grown under ambient oxygen conditions. We also show that the microenvironment within

the

C. albicans

biofilm is sufficient to support the growth of two different anaerobic species,

C. perfringens

and

B. fragilis

, and it is likely that decreased oxygen concentration plays a

major role in anaerobe survival. Different strains of

C. perfringens

and

B. fragilis

have been

reported to grow in oxygen levels as high as 3–5% (~40–70 μM) [

], and we have

shown that

C. albicans

biofilms provide an environment where the oxygen concentration is

as low as ~50 μM. This finding suggests that

C. albicans

may permit the growth of

anaerobes in oxic areas of the host that would otherwise be uninhabitable by those species.

This idea may be especially important for the establishment of

C. perfringens

infection,

which causes a wide variety of illnesses, including enterotoxemia, gas gangrene, and wound

infections, many of which are life-threatening [

The fact that oxygen concentration decreases steadily from the top to the bottom of a

albicans

biofilm adds to our understanding of the heterogeneous nature of biofilms.

albicans

biofilms are composed of multiple cell types (yeast, pseudohyphae, hyphae,

persister/dormant cells and dispersing cells) that express different genetic programs [

–

]

Fox et al.

Page 4

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

due to their precise location within the biofilm. The oxygen concentration gradient is one

critical variable that structures the biofilm microenvironment and suggests that metabolism

and gene expression vary between cells at different levels throughout the biofilm.

Partial induction of the white/opaque switch program in

C. albicans

We monitored the transcriptional response of

C. albicans

to bacterial species in mixed

biofilms, and found there was significant overlap between the genes upregulated by co-

culture with

K. pneumoniae

and genes enriched in opaque cells compared to white cells (p =

8.4×10

−20

). There is also significant overlap between genes upregulated by co-culture with

K. pneumoniae

and genes enriched in a strain overexpressing

WOR1

after passage through

the mouse gut, compared to a wild type strain (p = 3.4×10

−9

) [

]. We propose that

induction of

WOR1

by bacteria may prime

C. albicans

for white-opaque switching, but that

additional environmental cues are needed to fully induce the switch to the opaque form. An

alternative hypothesis is that partial induction of the opaque program is an adaptive response

to exposure to bacteria.

Aggregation induction by co-culture in suspension

We found that

C. perfringens

induces aggregation of

C. albicans

in ambient oxic suspension

cultures and that the aggregates, which contain both fungi and bacteria, allow

C. perfringens

to survive in a normally toxic environment. Induction of aggregation may be similar to

induction of biofilm formation, as aggregation requires the same master regulators needed

for

C. albicans

to form a “conventional” biofilm on a solid surface. Moreover, the cells in

the aggregates resemble cells in biofilms on solid surfaces. These observations indicate that

the biofilm “program” in

C. albicans

does not require a solid surface to become activated,

and the definition of a

C. albicans

biofilm may be expanded from a substrate-attached

community to include suspended aggregates.

E. coli, Pediococcus damnosus

, and several

other bacterial species were previously found to induce aggregation when co-cultured with

several yeast species, including

Candida utilis, S. cerevisiae,

and

Schizosaccharomyces

pombe

[

]. The evidence suggests that many microbial species are able to co-aggregate,

and our work has demonstrated that adherence between fungi and bacteria can allow the

survival of the bacteria.

Interspecies Interactions

We have shown that

C. albicans

interacts in a variety of ways with several representative

species of the gut microbiome. These microbes are clearly able to sense one another; for

example

C. albicans

responds through large changes in adherence and gene expression. We

have provided new evidence of antagonistic (reduction of

C. albicans

biofilm thickness by

the presence of

K. pneumoniae

) and beneficial (protection of

C. perfringens

C. albicans

biofilms) relationships, and have begun to uncover the genes involved in these interactions.

These findings highlight the importance of considering the microenvironments encountered

by microbiome members. The strategy of studying pairwise interactions between fungi and

bacteria in the context of heterogeneous microenvironments can be expanded to better

understand the complex community of thousands of species that encounter one another in

the host.

Fox et al.

Page 5

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

Experimental Procedures

Co-cultures in suspension or biofilms

C. albicans

and/or bacteria were grown in suspension or in biofilms adhered in 6-well

polystyrene plates, in Brain Heart Infusion (BHI) medium, supplemented with 5% fetal

bovine serum (BHI-FBS). Additional details in Supplement.

Colony Forming Units (CFUs) Assay

CFUs were plated from serial dilutions of either biofilms or suspension cultures. Dilutions

were plated on YPD agar, LB agar, or blood agar at 30°C or 37°C, depending on the species.

Additional details in Supplement.

Oxygen measurement

Oxygen concentration in biofilms was measured with a Unisense STOX-Sensor

microelectrode, with measurements obtained every 10 μm from top to bottom. Additional

details in Supplement.

Gene expression microarrays

Cy3 or Cy5-labeled cDNA was hybridized to custom Agilent microarrays, analyzed in

GenePix Pro, and normalized with LOWESS. Additional details in Supplement.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank Matthew Lohse, Aaron Hernday, Chiraj Dalal, Oliver Homann and Jose Christian Perez for strains or

plasmids used in this study, Sheena Singh Babak and Trevor Sorrells for comments on the manuscript, and Jorge

Mendoza for technical assistance. We appreciate use of the UCSF Nikon Imaging Center. This study was supported

by National Institutes of Health (NIH) grant R01AI083311 (A.D.J.) and by a UCSF Program for Breakthrough

Biomedical Research award, funded partly by the Sandler Foundation. E.P.F. was supported by NIH fellowship

T32AI060537, C.J.N. was supported by NIH grant K99AI100896, and D.K.N. was supported by the Howard

Hughes Medical Institute (HHMI) and the National Heart, Lung, and Blood Institute of the NIH (R01HL117328).

D.K.N. is an HHMI Investigator.

References

1. Savage DC. Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol. 1977; 31:107–33.

[PubMed: 334036]

2. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE,

Relman Da. Diversity of the human intestinal microbial flora. Science. 2005; 308:1635–8.

[PubMed: 15831718]

3. López D, Vlamakis H, Kolter R. Biofilms. Cold Spring Harb Perspect Biol. 2010; 2:a000398.

[PubMed: 20519345]

4. Kolter R, Greenberg EP. Microbial sciences: the superficial life of microbes. Nature. 2006;

441:300–2. [PubMed: 16710410]

5. Donlan RM, Costerton JW. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms.

2002; 15:167–193.

Fox et al.

Page 6

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

6. Wolcott R, Costerton JW, Raoult D, Cutler SJ. The polymicrobial nature of biofilm infection.

2012:1–6.

7. Iliev ID, Funari Va, Taylor KD, Nguyen Q, Reyes CN, Strom SP, Brown J, Becker Ca, Fleshner PR,

Dubinsky M, et al. Interactions between commensal fungi and the C-type lectin receptor Dectin-1

influence colitis. Science. 2012; 336:1314–7. [PubMed: 22674328]

8. Moyes DL, Naglik JR. The mycobiome: influencing IBD severity. Cell Host Microbe. 2012;

11:551–2. [PubMed: 22704612]

9. Ghannoum, Ma; Jurevic, RJ.; Mukherjee, PK.; Cui, F.; Sikaroodi, M.; Naqvi, A.; Gillevet, PM.

Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog.

2010; 6:e1000713. [PubMed: 20072605]

10. Khatib R, Riederer KM, Ramanathan J, Baran J. Faecal fungal flora in healthy volunteers and

inpatients. Mycoses. 2001; 44:151–6. [PubMed: 11486452]

11. Nobile CJ, Mitchell AP. Regulation of cell-surface genes and biofilm formation by the C. albicans

transcription factor Bcr1p. Curr Biol. 2005; 15:1150–5. [PubMed: 15964282]

12. Nobile CJ, Fox EP, Nett JE, Sorrells TR, Mitrovich QM, Hernday AD, Tuch BB, Andes DR,

Johnson AD. A recently evolved transcriptional network controls biofilm development in Candida

albicans. Cell. 2012; 148:126–38. [PubMed: 22265407]

13. Revsbech NP, Larsen LH, Gundersen J, Dalsgaard T, Ulloa O, Thamdrup B. Determination of

ultra-low oxygen concentrations in oxygen minimum zones by the STOX sensor. Limnol

Oceanogr Methods. 2009; 7:371–381.

14. Slutsky B, Staebell M, Anderson J, Risen L, Pfaller M, Soll DR. “White-opaque transition”: a

second high-frequency switching system in Candida albicans. J Bacteriol. 1987; 169:189–97.

[PubMed: 3539914]

15. Srikantha T, Borneman AR, Daniels KJ, Pujol C, Wu W, Seringhaus MR, Gerstein M, Yi S,

Snyder M, Soll DR. TOS9 regulates white-opaque switching in Candida albicans. Eukaryot Cell.

2006; 5:1674–87. [PubMed: 16950924]

16. Zordan RE, Galgoczy DJ, Johnson AD. Epigenetic properties of white-opaque switching in

Candida albicans are based on a self-sustaining transcriptional feedback loop. Proc Natl Acad Sci

U S A. 2006; 103:12807–12. [PubMed: 16899543]

17. Rikkerink EH, Magee BB, Magee PT. Opaque-white phenotype transition: a programmed

morphological transition in Candida albicans. J Bacteriol. 1988; 170:895–9. [PubMed: 2828333]

18. Lohse MB, Johnson AD. Temporal anatomy of an epigenetic switch in cell programming: the

white-opaque transition of C.albicans. Mol Microbiol. 2010; 78:331–43. [PubMed: 20735781]

19. Hernday AD, Lohse MB, Fordyce PM, Nobile CJ, DeRisi JL, Johnson AD. Structure of the

transcriptional network controlling white-opaque switching in Candida albicans. Mol Microbiol.

2013; 90:22–35. [PubMed: 23855748]

20. Tuch BB, Mitrovich QM, Homann OR, Hernday AD, Monighetti CK, De La Vega FM, Johnson

AD. The transcriptomes of two heritable cell types illuminate the circuit governing their

differentiation. PLoS Genet. 2010; 6:e1001070. [PubMed: 20808890]

21. Zordan RE, Miller MG, Galgoczy DJ, Tuch BB, Johnson AD. Interlocking transcriptional

feedback loops control white-opaque switching in Candida albicans. PLoS Biol. 2007; 5:e256.

[PubMed: 17880264]

22. Synnott JM, Guida A, Mulhern-Haughey S, Higgins DG, Butler G. Regulation of the hypoxic

response in Candida albicans. Eukaryot Cell. 2010; 9:1734–46. [PubMed: 20870877]

23. Homann OR, Dea J, Noble SM, Johnson AD. A phenotypic profile of the Candida albicans

regulatory network. PLoS Genet. 2009; 5:e1000783. [PubMed: 20041210]

24. Martin R, Moran GP, Jacobsen ID, Heyken A, Domey J, Sullivan DJ, Kurzai O, Hube B. The

Candida albicans-specific gene EED1 encodes a key regulator of hyphal extension. PLoS One.

2011; 6:e18394. [PubMed: 21512583]

25. Zhao X, Daniels KJ, Oh S, Green CB, Yeater KM, Soll DR, Hoyer LL. Candida albicans Als3p is

required for wild-type biofilm formation on silicone elastomer surfaces. Microbiology. 2006;

152:2287–99. [PubMed: 16849795]

26. Liu Y, Filler SG. Candida albicans Als3, a multifunctional adhesin and invasin. Eukaryot Cell.

2011; 10:168–73. [PubMed: 21115738]

Fox et al.

Page 7

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

27. Silverman RJ, Nobbs AH, Vickerman MM, Barbour ME, Jenkinson HF. Interaction of Candida

albicans cell wall Als3 protein with Streptococcus gordonii SspB adhesin promotes development

of mixed-species communities. Infect Immun. 2010; 78:4644–52. [PubMed: 20805332]

28. Peters BM, Ovchinnikova ES, Krom BP, Schlecht LM, Zhou H, Hoyer LL, Busscher HJ, van der

Mei HC, Jabra-Rizk MA, Shirtliff ME. Staphylococcus aureus adherence to Candida albicans

hyphae is mediated by the hyphal adhesin Als3p. Microbiology. 2012; 158:2975–86. [PubMed:

22918893]

29. Nobile CJ, Andes DR, Nett JE, Smith FJ, Yue F, Phan QT, Edwards JE, Filler SG, Mitchell AP.

Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo.

PLoS Pathog. 2006; 2:e63. [PubMed: 16839200]

30. Bradshaw DJ, Marsh PD, Allison C, Schilling KM. Effect of oxygen, inoculum composition and

flow rate on development of mixed-culture oral biofilms. Microbiology. 1996; 142(Pt 3):623–9.

[PubMed: 8868437]

31. Bradshaw DJ, Marsh PD, Watson GK, Allison C. Role of Fusobacterium nucleatum and

coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during

aeration. Infect Immun. 1998; 66:4729–32. [PubMed: 9746571]

32. Rossignol T, Ding C, Guida A, D’Enfert C, Higgins DG, Butler G. Correlation between biofilm

formation and the hypoxic response in Candida parapsilosis. Eukaryot Cell. 2009; 8:550–9.

[PubMed: 19151323]

33. Bonhomme J, Chauvel M, Goyard S, Roux P, Rossignol T, D’Enfert C. Contribution of the

glycolytic flux and hypoxia adaptation to efficient biofilm formation by Candida albicans. Mol

Microbiol. 2011; 80:995–1013. [PubMed: 21414038]

34. Stichternoth C, Ernst JF. Hypoxic adaptation by Efg1 regulates biofilm formation by Candida

albicans. Appl Environ Microbiol. 2009; 75:3663–72. [PubMed: 19346360]

35. Loesche WJ. Oxygen sensitivity of various anaerobic bacteria. Appl Microbiol. 1969; 18:723–7.

[PubMed: 5370458]

36. Tally FP, Stewart PR, Sutter VL, Rosenblatt JE. Oxygen tolerance of fresh clinical anaerobic

bacteria. J Clin Microbiol. 1975; 1:161–4. [PubMed: 1176601]

37. Li J, Adams V, Bannam TL, Miyamoto K, Garcia JP, Uzal Fa, Rood JI, McClane Ba. Toxin

plasmids of Clostridium perfringens. Microbiol Mol Biol Rev. 2013; 77:208–33. [PubMed:

23699255]

38. Stevens DL, Aldape MJ, Bryant AE. Life-threatening clostridial infections. Anaerobe. 2012;

18:254–9. [PubMed: 22120198]

39. Lewis K. Persister cells. Annu Rev Microbiol. 2010; 64:357–72. [PubMed: 20528688]

40. Yeater KM, Chandra J, Cheng G, Mukherjee PK, Zhao X, Rodriguez-Zas SL, Kwast KE,

Ghannoum Ma, Hoyer LL. Temporal analysis of Candida albicans gene expression during biofilm

development. Microbiology. 2007; 153:2373–85. [PubMed: 17660402]

41. Uppuluri P, Chaturvedi AK, Srinivasan A, Banerjee M, Ramasubramaniam AK, Köhler JR,

Kadosh D, Lopez-Ribot JL. Dispersion as an important step in the Candida albicans biofilm

developmental cycle. PLoS Pathog. 2010; 6:e1000828. [PubMed: 20360962]

42. Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida albicans biofilms. J

Med Microbiol. 1999; 48:671–9. [PubMed: 10403418]

43. Nett JE, Lepak AJ, Marchillo K, Andes DR. Time course global gene expression analysis of an in

vivo Candida biofilm. J Infect Dis. 2009; 200:307–13. [PubMed: 19527170]

44. Pande K, Chen C, Noble SM. Passage through the mammalian gut triggers a phenotypic switch

that promotes Candida albicans commensalism. Nat Genet. 2013:1–6. [PubMed: 23268125]

45. Peng X, Sun J, Iserentant D, Michiels C, Verachtert H. Flocculation and coflocculation of bacteria

by yeasts. Appl Microbiol Biotechnol. 2001; 55:777–81. [PubMed: 11525628]

Fox et al.

Page 8

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

Highlights

•

C. albicans

biofilms are hypoxic and support anaerobic bacteria survival

•

Bacteria induce part of the

C. albicans

opaque genetic program in mixed

biofilms

•

C. perfringens

induces biofilm formation in

C. albicans

in suspension co-culture

Fox et al.

Page 9

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

Figure 1.

C. albicans

forms biofilms with five different species of bacteria

in vitro

C. albicans

was grown in biofilms for 24 h either alone (A), or with

E. coli

(B),

pneumoniae

(C),

E. faecalis

(D),

C. perfringens

(E), or

B. fragilis

(F). Biofilms were stained

with conconavalin A – Alexa 594 and Syto 13 dyes, then imaged by CSLM. Images are

maximum intensity projections of the top and side view. Representative images of at least

three replicates are shown. Scale bars are 50 μm. See also Figure S1.

Fox et al.

Page 10

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

Figure 2. Mixed-species biofilms provide a niche for growth of anaerobic bacteria

(A–D) CFU/ml of indicated species grown in biofilms in monoculture or co-culture under

oxic or anoxic conditions. Cells were collected from biofilms only (not from the media

above the biofilm) at 1.5, 4, 24, 48, and 72 h, and plated for CFUs. A)

C. albicans

and/or

perfringens

in oxic conditions. B)

C. albicans

and/or

B. fragilis

in oxic conditions. C)

albicans

and/or

C. perfringens

in anoxic conditions. D)

C. albicans

and/or

B. fragilis

anoxic conditions. E) Oxygen was measured in biofilms composed of the indicated species

using a STOX-Sensor. Readings were taken every 10 μm from the top to the bottom of the

biofilm. For all graphs, the mean of at least two replicates is shown, with error bars

representing standard deviation. See also Figure S2.

Fox et al.

Page 11

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

Figure 3. Co-culture with bacteria in biofilms induces differential gene expression in

C. albicans

A) Heat map of gene expression in

C. albicans

when co-cultured with the indicated species

in biofilms, compared to

C. albicans

alone. Shown are the median values of at least two

biological replicates. Control refers to

C. albicans

with media added to mimic the inoculum

with bacteria, compared to

C. albicans

alone. 2863 genes differentially regulated at least

twofold in at least one condition are displayed along the x-axis. Upregulated genes are

yellow, downregulated genes are blue. B) Gene expression pattern of genes encoding

transcription regulators that control the white-opaque switch circuit. The top panel shows

expression levels measured in opaque vs. white cells from [

]. The bottom panel shows

expression levels when

C. albicans

is co-cultured in biofilms with the indicated bacterial

species, compared to

C. albicans

alone. See also Figure S3.

Fox et al.

Page 12

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript

Figure 4.

C. perfringens

induces aggregation of

C. albicans

during ambient oxic, suspension co-

culture

Suspension cultures of

C. albicans

with or without

C. perfringens

, grown for 4 h or 24 h at

37°C, in anoxic or ambient oxic conditions. A–F) 4 h growth. A)

C. albicans

alone, anoxic.

C. albicans

C. perfringens

, anoxic. C)

C. albicans

alone, oxic. D)

C. albicans

perfringens

, oxic. E)

C. albicans

+ cell-free supernatant from

C. perfringens

culture. F)

albicans

+ heat-killed

C. perfringens

cells. G)

C. albicans

and/or

C. perfringens

imaged by

light microscopy. Representative images are shown. Scale bars are 20 μm. H–I) CFU/ml of

indicated species grown in monoculture or co-culture, in suspension cultures under ambient

oxic or anoxic conditions. H)

C. albicans

and/or

C. perfringens

in ambient oxic conditions.

C. albicans

and/or

C. perfringens

in anoxic conditions. Shown is the mean of at least two

replicates, error bars are standard deviation. J–R)

C. albicans

wild type or mutant strains

grown in suspension, in ambient oxygen, for 4 h with

C. perfringens.

J) WT. K)

rim101

Δ/Δ.

flo8

Δ/Δ. M)

brg1

Δ/Δ. N)

tec1

Δ/Δ. O)

rob1

Δ/Δ. P)

bcr1

Δ/Δ. Q)

efg1

Δ/Δ. R)

ndt80

Δ/Δ.

Assay was performed at least twice for each condition or mutant strain. See also Figure S4.

Fox et al.

Page 13

Curr Biol

. Author manuscript; available in PMC 2015 October 20.

NIH-PA Author Manuscript