FEMS Yeast Research, 15, 2015, fov081
doi: 10.1093/femsyr/fov081
Advance Access Publication Date: 7 September 2015
Minireview
MINIREVIEW
Candida albicans commensalism in the
gastrointestinal tract
B. Anne Neville1,2,† , Christophe d’Enfert1,2,∗
and Marie-Elisabeth Bougnoux1,2,3
1
Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Mycologie, F-75015 Paris, France,
INRA, USC2019, F-75015 Paris, France and 3 Laboratoire de Parasitologie-Mycologie, Service de Microbiologie,
Hôpital Necker-Enfants Malades, Université Paris Descartes, Faculté de Médicine, F-75015 Paris, France
2
∗ Corresponding author: Unité Biologie et Pathogénicité Fongiques, 25-28 rue du Docteur Roux, Institut Pasteur, 75724 Paris Cedex 15, France.
Tel: +33140613257; Fax: +33140613456; E-mail: [email protected]
†
Present address: Host-Microbiota Interactions Laboratory, Wellcome Trust Sanger Institute, Hinxton, UK
One sentence summary: Major progress in understanding the tripartite interaction involving the fungal pathogen of humans, Candida albicans, the
gastrointestinal microbiota and host immunity has been made recently.
Editor: Carol Munro
ABSTRACT
Candida albicans is a polymorphic yeast species that often forms part of the commensal gastrointestinal mycobiota of
healthy humans. It is also an important opportunistic pathogen. A tripartite interaction involving C. albicans, the resident
microbiota and host immunity maintains C. albicans in its commensal form. The influence of each of these factors on
C. albicans carriage is considered herein, with particular focus on the mycobiota and the approaches used to study it,
models of gastrointestinal colonization by C. albicans, the C. albicans genes and phenotypes that are necessary for
commensalism and the host factors that influence C. albicans carriage.
Keywords: Candida albicans; commensalism; microbiota; mycobiota; gastrointestinal tract; animal models; immunity
GENERAL INTRODUCTION
Candida albicans is a commensal yeast species that is found in
the gastrointestinal (GI) tracts of humans and other animals.
Vertical transmission of this microorganism from mother to
infant during birth (Waggoner-Fountain et al. 1996; BlaschkeHellmessen 1998) means that humans often form a life-long association with this species. Nevertheless, this host–microbe relationship is not always benign, and infections with C. albicans
among ‘at risk’ cohorts bear considerable morbidity and mortality (Brown et al. 2012). Thus, as might be expected for a prevalent pathobiont, the capacity of C. albicans for both commensalism and pathogenesis and the transition between these lifestyle
options are the focus of much ongoing research. In this review,
we discuss the fungal component of the GI microbiota of humans, with a particular focus on C. albicans and its interaction
with the rest of the microbiota and its host. We outline the approaches used to study commensalism and dissemination by
this yeast in animal models, focusing in particular, on the recent research describing C. albicans commensalism (without an
eventual transition to infection) in the GI tract. We describe
the various C. albicans morphologies and the genes required for
commensalism in mouse models. We also discuss the host response to C. albicans in the GI tract, describing the immune players involved, and we detail the inconsistencies of the emergent
research in this area.
Received: 3 June 2015; Accepted: 26 August 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Yeast Research, 2015, Vol. 15, No. 7
THE FUNGAL COMPONENT OF THE GI
MICROBIOTA—THE MYCOBIOTA
The complete human gut microbiota includes microorganisms
from each of the principal microbial lineages: bacteria, archaea,
eukaryota and viruses (including bacteriophage) (Norman,
Handley and Virgin 2014). The term ‘microbiota’ however, while
formally intended to describe the totality of the microorganisms
present in a particular environment (Medical Subject Headings
www.ncbi.nlm.nih.gov/mesh), has often in practice been used to
refer exclusively to the bacterial component of any given ecosystem. The widespread use of bacterial 16S rRNA gene targets to
describe and evaluate ‘microbiota’ (without a ‘bacterial’ qualifier) composition is testament to the more selective usage of
this word in the scientific literature. In contrast, the term ‘mycobiota’, which is derived from the Greek words myco (fungus)
and bios (life), can be unambiguously used to refer exclusively to
the fungal community of any given habitat. Moreover, the term
‘microbiome’ has an indiscriminate and all-inclusive sense, and
shotgun metagenomics studies of the human gut microbiome
(the DNA of all organisms present in a particular niche) have also
yielded some insight into the fungal community present.
Usage of ITS, the universal fungal DNA barcode for
high-throughput amplicon sequencing, lacks
standardized implementation
The internal transcribed spacer (ITS) regions of the ribosomal
DNA are the preferred molecular targets for compositional analysis of the mycobiota by high-throughput amplicon sequencing
(Schoch et al. 2012; Bates et al. 2013). In fungal genomes, the
genes encoding the 18S, 5.8S and 25S ribosomal RNA subunits
are found contiguously in multicopy, and are separated from
each other by stretches of DNA of variable length—the ITSs. Two
such regions, ITS-1 and ITS-2, separate the three genes from
each other. The ITS regions are intrinsically variable in length,
ranging from ∼450 bp to ∼700 bp (Bellemain et al. 2010). Thus,
any given set of standard PCR oligonucleotides will yield amplicons of different sizes from different fungal species (Bellemain
et al. 2010; Bokulich and Mills 2013). This creates an inherent
PCR bias, because short products are likely to be preferentially
amplified.
A scientific consensus has not yet been established for the
precise ITS region to target for high-throughput amplicon sequencing. Some authors have preferred amplification of the entire ITS region (ITS-1, 5.8S gene, ITS-2), while others have targeted either ITS-1 or ITS-2 (LaTuga et al. 2011; David et al. 2014).
While the ITS-1 region is recommended for high-throughput sequencing (Bokulich and Mills 2013), the ITS-2 region is advocated for species in which spliceosomal inserts near the 3 end
of the 18S rRNA gene would bias amplification of ITS-1 (Bates
et al. 2013). Furthermore, the target amplicon size should be
matched to an appropriate sequencing technology to maximize
the phylogenetic signal that can be obtained through sequencing. For example, long amplicons, such as those encompassing
ITS-1, 5.8S rRNA and ITS-2, can be successfully sequenced using
454-pyrosequencing technology, thereby maximizing signal for
taxon assignment and phylogenetic analysis (Bokulich and Mills
2013). It has also been recommended to prepare multiple PCR
reactions for each environmental DNA sample, some of which
would target different ITS regions, and to perform the reactions
at more than one annealing temperature, to overcome amplification biases (Diakarya vs non-Diakarya, Ascomycota vs Basid-
iomycota), which would otherwise confound a reliable community analysis (Bellemain et al. 2010).
The utility of community profiling by modern highthroughput sequencing methods is also highly dependent on
researchers’ ability to assign the sequenced reads to a taxon
of defined nomenclature. Without the public availability of
well-curated and up-to-date databases which include annotated fungal ITS (and for metagenome data, fungal genome
sequences) for specific species, large proportions of generated
sequence data would remain unclassified. While a number of
sequence databases exist for fungal taxonomy assignment,
[UNITE (Abarenkov et al. 2010) (https://unite.ut.ee/), SILVA
(Pruesse et al. 2007) (http://www.arb-silva.de/), MaarjAM (Opik
et al. 2010) (http://maarjam.botany.ut.ee/)], the UNITE database
is recommended for ITS-based taxon assignment while SILVA
is appropriate for SSU-based targets (Lindahl et al. 2013). Nevertheless, it has been estimated that less than 5% of fungal
species that are thought to exist on earth are represented in
databases (Kõljalg et al. 2013). To address the problem of sequence reads for which no meaningful taxonomic information
is available, the UNITE team has introduced the concept of the
‘species hypothesis’ (SH). A ‘species hypothesis’ is a group of
two or more sequence reads which have been clustered to the
approximate species level subsequent to an initial clustering to
the subgenus/genus level, though singletons are also tolerated.
If an expert-specified reference sequence has not been defined,
a representative sequence for the new SH is designated and
is assigned a unique SH number, which allows the sequence
to be classified. The value of this approach is that it allows
researchers to approximate the taxon assignment of sequence
reads for unknown fungi and serves as a standardized reference
for that particular sequence over time (Kõljalg et al. 2013).
Limitations in our understanding of the human
intestinal mycobiota
The paucity of information in the scientific literature regarding the eukaryotic component of the intestinal microbiota of
healthy humans is a problem that has been acknowledged
for several years. An early culture-independent study of the
intestinal mycobiota of healthy adults targeted the eukaryotic 18S rDNA gene and the fungal ITS region (Scanlan and
Marchesi 2008). Rather than using a high-throughput sequencing approach (a method that has only quite recently become
widely available), traditional methods for community profiling
by molecular methods, namely denaturing gel gradient electrophoresis and clone library preparation followed by Sanger sequencing, were used. As such, the sampling depth was probably quite limited. The overall diversity of eukaryotes was low,
and fungal sequences were detected in ∼82% (14/17) of the individuals tested. Based on a Fungal Internal Transcribed Spacer
Analysis, fungal communities were deemed relatively stable
over time, although allochthonous fungal species (those that are
transiently present and which were not detected at every sampling occasion) were also detected. Such species include foodassociated fungi, such as Penicillium roqueforti, which is found in
certain cheeses. In contrast, species of the genera Galactomyces,
Paecilomyces and Gloeotinia, some of which may also be foodborne fungi, were present at all time points and were suggested
as common and stable inhabitants of the human GI tract (Scanlan and Marchesi 2008). Furthermore, there was an incongruence between the results of the culture-dependent and cultureindependent analyses of the fungal communities in these individuals, emphasizing the limitations and biases of each of these
Neville et al.
experimental approaches for comprehensive community profiling and assessment.
More recently, a meta-analysis of the ‘mycobiota’ literature,
which focused on 75 research articles published in English between 1998 and 2014, provided a list of the fungal genera and
species that occur in healthy individuals and those that were
detected in individuals with a particular disease (Gouba and
Drancourt 2015). The intestinal mycobiota of healthy individuals was reported to include 247 fungal species, which belong to
the phyla Ascomycota (63%), Basidiomycota (32%), Zygomycota
(3%) and non-classified fungi (2%) (Gouba and Drancourt 2015).
These authors also acknowledge the discrepancies in the number of fungal species that can be identified by culture-dependent
versus culture-independent techniques. They also indicate that
the fungal community profile varies depending on the body site
sampled, with the fungal communities of the oral and intestinal mycobiotas being different and with little overlap in species
membership.
The application of modern, high-throughput sequencing and
data analysis methods and the continuing improvement of reference databases thus provide a means to address the dearth of
information on the GI mycobiota of healthy adult humans. The
outcomes of several recent studies which availed of these technologies for mycobiota profiling will now be discussed.
Insights into the human intestinal mycobiota from
high-throughput amplicon sequencing
A pyrosequencing-based study of the eukaryotic component of
the fecal microbiota of eight healthy adult humans revealed
that fungi accounted for most of the operational taxonomic
units (OTUs) identified, whether assessed by 18S rRNA (∼62%) or
fungal-specific ITS (∼90.5%) gene amplicons (Dollive et al. 2012).
The ITS sequences were assigned to 215 OTUs, while the 18S
rRNA gene-derived reads yielded 93 OTUs. Nevertheless, most
of the reads were assigned to few OTUs, implying that the taxa
detected were present in unequal amounts. Ascomycota and Basidiomycota accounted for 81 and 17.2% of the reads from 18S
rRNA gene-derived sequences, respectively while these phyla
were represented at 57.4 and 25.7% respectively in the ITS data.
The low number of food and plant reads among the sequenced
amplicons resulted from a deliberate effort to minimize signal
from mammalian and dietary plant sources through prudent
primer design. Interestingly, 37% more reads were returned from
the 18S rRNA gene amplicons than from the ITS-derived products, demonstrating how the choice of molecular target region
and primer combinations can impact the outcome of mycobiota
analyses.
The influence of diet and microbiota composition on the
diversity of the fecal fungal and archaeal communities was
studied using stool collected from 96 healthy adult humans
(Hoffmann et al. 2013). Each individual tested harbored a fungal
community that was dominated at the phylum level by either
the Ascomycota or the Basidiomycota. Substantial interindividual variation in the composition of the fungal community at the
genus level was observed, with only 12 of the 66 fungal genera
identified being common to 9 or more of the individuals tested.
Saccharomyces and Candida were the most prevalent of these genera, being detected in 89 and 57% of the subjects respectively.
While the prevalence of Saccharomyces reads could derive from
food-associated consumption of this species (via bread or beer),
the relative abundance of the genus Candida was found to be significantly influenced by an individual’s short-term diet. The relative abundance of the genus Candida was positively correlated
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with carbohydrate consumption, but negatively correlated with
the ingestion of saturated fatty acids (Hoffmann et al. 2013).
The structure of the microbiota was examined by investigating the relationships between the fungi, bacteria and archaea present. The genus Candida tended to cooccur (Dice index > 0.5) with the bacterial genera Faecalibacterium, Roseburia
and Parabacteroides, the fungal genus Saccharomyces and with uncharacterized species of the bacterial families Lachnospiraceae
and Ruminococcaceae. The genus Candida was found to significantly and positively covary with the bacterial genera
Xylanibacter, Catenibacterium and a member of the family Prevotellaceae. It also cooccurred (Dice index 0.61), but negatively
covaried with the genus Bacteroides (Bacteria). Candida was found
to be positively and negatively associated with Methanobrevibacter (Archaea) and Nitrososphaera (Archaea), respectively.
Furthermore, it was the proportion, rather than the types
of microbes present which influenced microbiota composition.
For example, the Prevotella/Bacteroides ratio correlated with the
amount, but not the type, of fungi present. Similarly, significant
correlations between the phyla Ascomycota and Basidiomycota
and various bacterial taxa were returned only when taxon abundance and not simply taxon diversity was considered (Hoffmann
et al. 2013).
A study of 10 American adults who volunteered to follow either a plant- or animal- based dietary regime over 5-day period
also revealed that the size and composition of the enteric fungal
community is directly impacted by host diet (David et al. 2014).
Cured meats and several cheese products were found to harbor
a fungal community consisting of Candida species and members of the fungal genera, Debaryomyces, Penicillium and Scopulariopsis. Furthermore, diets rich in animal-derived products
supported a significant increase in the concentration of viable
fungi in feces relative to baseline levels (before dietary intervention) (David et al. 2014). Like in the study described above (Hoffmann et al. 2013), Candida species became more abundant on the
carbohydrate-rich plant diet. These analyses (Hoffmann et al.
2013; David et al. 2014), however, are limited to a discussion of
the effect of diet on the genus Candida, rather than on C. albicans
or any other Candida species specifically.
Thus, high-throughput amplicon sequencing provides considerable insight into the fungal communities of the human gut.
The composition of the GI mycobiota is influenced both by ingested fungal species and the interaction of commensal fungi
with the rest of the gut microbiota. Nevertheless, despite these
initial studies, the autochthonous species of the healthy human
gut mycobiota have not been well described and cannot at this
time be distinguished from the allochthonous (most likely foodborne) fungal species transiting this niche.
Moreover, the technical choices made when isolating DNA
and when selecting PCR oligonucleotides and a sequencing platform could have a considerable impact on the results. Standardization of protocols to establish best practice procedures is
needed if mycobiota profiling by high-throughput sequencing
methods is to become routine (Bates et al. 2013; Lindahl et al.
2013).
Insights into the human intestinal mycobiota from
shotgun metagenomics studies
Metagenomics studies of the human gut microbiota have rarely
focused on the functions contributed by the non-bacterial
microbes present. For the eukaryotic community specifically,
there may be several reasons for this. First, the non-bacterial
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FEMS Yeast Research, 2015, Vol. 15, No. 7
community is overall quite small, and fungi comprise only a
fraction of this. For example, the Metahit consortium (Arumugam et al. 2011) identified eukaryotic DNA fragments in their
metagenome data at a low level (<1.3% of DNA from any sample;
0.5% on average, based on homology to eukaryotic sequences in
the STRING database, version 8). Together, fungi and metazoa
accounted for the largest average fraction of the eukaryotic sequences (∼0.3%), with fungi specifically contributing an average
of 0.11% of these (Arumugam et al. 2011).
Furthermore, eukaryotic genomes are often large, with a low
coding density (Wood et al. 2002). Consequently, a considerably
greater depth of sequencing would be required to access the genetic content of the eukaryotic community at a level comparable
to that routinely obtained for the prokaryotic community, particularly in niches harboring a diverse fungal community. This
is exemplified in a non-redundant microbial gene catalogue representing the fecal microbiota of 124 Europeans, in which only
∼0.1% of the genes were of eukaryotic origin while 99.1% of the
genes were derived from bacteria (Qin et al. 2010). The additional
sequencing that would be required to achieve a depth sufficient
to access fungal genes is likely to be prohibitive, rendering an
analysis of the specific contributions of the intestinal eukaryotic community beyond the scope of most metagenome studies
performed to date.
Nevertheless, with the falling cost of sequencing combined
with the greater availability of curated fungal genomes in publically accessible reference databases, and the development of the
necessary tools and expertise which will enable labs to handle
and analyse their sequencing data ‘in house’, we can expect that
metagenomics studies will incorporate more detailed descriptions of the contributions of the fungal and archaeal communities in the future. One such initiating metagenome project provides insight into the intestinal mycobiota on an international
scale.
In a comparative study of the intestinal microbiomes of infants and adults from Malawi, the United States and Amerindians from Venezuela (Yatsunenko et al. 2012), in which a total
of 5.9 Gb of sequencing data was generated, only 7 ± 8% of the
reads representing 110 metagenomes, could be mapped to nonbacterial sequences. The majority of these non-bacterial reads
corresponded to either archaea or fungi. For each human population examined, the fungal proportion of the microbiota was
always significantly greater in adults than in children. Furthermore, the microbiomes of individuals from the USA contained
significantly fewer fungal sequences than those of either the
Malawian or Venezuelan adults. These differences in mycobiota
size and structure could possibly reflect differences in the diets
of adults and children within and across the different societies.
Sequences of the phyla Ascomycota and Microsporidia were the
most abundant fungal sequences in each of the three populations tested (Yatsunenko et al. 2012).
Thus, the burgeoning field of ‘gut microbiomics’ has not yet
well integrated the resident and transient fungi nor the nonprokaryotic microbes in general, though this is likely to change
with time. These hitherto neglected microbial populations cannot continue to go unnoticed. The microbiota–mycobiota dynamics in the gut are starting to be appreciated (Cuskin et al.
2015) and given their contribution to biomass in the gut (Underhill and Iliev 2014), enteric fungi are likely to bear significant
immunomodulatory potential.
MODELS OF GI COLONIZATION BY
C. ALBICANS
The study of the C. albicans—host relationship in the laboratory often relies on the development of appropriate in vivo
models that recapitulate the characteristics of either infection
or commensalism in humans. Although several models have
been developed for these purposes (Van Cutsem and Thienpont
1971; Hube et al. 1997; Braun et al. 2000; Schinabeck et al. 2004;
Hoeflinger et al. 2014), models involving mice have been most
commonly used to study C. albicans commensalism in the stomach and lower GI tract (Schofield et al. 2005; Koh 2013; Pérez,
Kumamoto and Johnson 2013; Prieto et al. 2014).
In general, the mouse is a desirable model organism because its small size means that large numbers of animals can
be housed and handled easily and inexpensively. Furthermore,
mouse models often recapitulate major characteristics of human diseases and the availability of methods to manipulate the
mouse genome means that researchers can generate or purchase mice of specific mutant genotypes of interest if necessary.
The establishment of rodent models of intestinal colonization
by C. albicans is, however, complicated by the fact that this yeast
does not naturally colonize these animals (Huppert, Cazin and
Smith 1955). The gut microbiota of laboratory rodents is quite
unlike that of humans (Loan et al. 2015), and the colonization
resistance that it imposes is sufficient to prevent the establishment of a large enteric C. albicans population. Thus, experimental interventions are required to facilitate stable intestinal colonization in these animals (Romani 2001; Clancy, Cheng and
Nguyen 2009; Koh 2013).
Antibiotic treatments have often been the intervention of
choice to establish long-term and stable colonization of the
murine gut by C. albicans. Advantages of this system include
the potential for the use of immunologically competent wildtype (WT) mice, the widespread and cheap availability of the
antimicrobial drugs, the high levels of colonization that can be
achieved with the system, the non-invasive dosing method (if
antibiotics are delivered via drinking bottles) and its simple setup, with no specific training or apparatus required. Disadvantages of the system include variation in the amount of antibiotic
solution consumed by the animals resulting in variable doses,
the possibility of wastage of antibiotic solutions, widespread
loss of microbiota structure following broad spectrum antibiotic treatment, antibiotic-associated diarrhoea as a side effect
of treatment and an unwanted impact on the immune system
or other physiological consequences of antibiotic exposure. Furthermore, dosages found to be effective in animals may not be
easily applied to humans.
While, genetic interventions centred on immunosuppression
(reviewed in Koh 2013) have been used for C. albicans colonization of the GI tract, methods that do not require antibiotic treatments or immunosuppression have also been reported. For example, the use of purified diets (Yamaguchi et al. 2005), regular
C. albicans dosage via food (Samonis et al. 1990, 2011), immunodeficient germ-free animals (Schofield et al. 2005) and the inoculation of mouse pups with C. albicans soon after birth may allow the establishment of a sizable C. albicans population in these
animals (reviewed in Romani 2001). In each case, however, it is
necessary to characterize the model to verify the absence of dissemination and disease.
Neville et al.
THE ROLE OF THE INTESTINAL MICROBIOTA
IN THE REGULATION OF ENTERIC C. ALBICANS
POPULATIONS
A tripartite interaction involving the host’s immune system,
the intestinal microbiota and C. albicans regulates the population size of this yeast in the gut. Evidence from animal models
(Zelante et al. 2013) and human trials (Romeo et al. 2011; Kumar
et al. 2013; Roy et al. 2014) has demonstrated that certain bacterial
populations can specifically restrain the outgrowth of C. albicans
in this niche. The identification of bacterial genera, species or
strains with anti-Candida properties could potentially be developed as probiotics against this yeast. Although certain products
of bacterial metabolism may offer generic anti-Candida properties, such as short-chain fatty acid production which has been
demonstrated to inhibit fungal growth and morphogenesis by
C. albicans in vitro (Noverr and Huffnagle 2004), bacteria may also
possess strain-specific anti-Candida properties. Several publications testify to the in vivo anti-C. albicans properties of various
bacteria.
One such study showed how the metabolism of dietary tryptophan by selected members of the stomach microbiota of mice
influenced colonization resistance towards C. albicans (Zelante
et al. 2013). Specifically, Lactobacillus species that harbored a gene
for an aromatic amino acid aminotransferase (araT), which enabled the bacterium to produce the indole metabolite, indole-3aldehyde (IAld) from dietary tryptophan, were able to stimulate
IL-22 production in the murine stomach and in specific immune
cells via the host aryl-hydrocarbon receptor (AhR). Although the
AraT enzyme is phylogenetically conserved, it is not present in
all bacterial lineages, and is absent from Clostridia species of
clusters IV and XIVa that are dominant in the murine gut. Thus,
not every species is capable of antagonizing C. albicans or of potentiating an antifungal effect in this manner.
Experiments in which antibiotics or dietary tryptophan were
used to modulate the indigenous Lactobacillus community in the
mouse stomach demonstrated how the C. albicans population of
intragastrically inoculated mice was correlated with the size and
composition of the gastric Lactobacillus population (Zelante et al.
2013). Colonization resistance towards Candida was increased
in WT mice during tryptophan feeding (promotes expansion of
the Lactobacillus community) and was decreased following ampicillin treatment (decreases the Lactobacillus community). In WT
mice consuming a diet rich in tryptophan, this anti-Candida resistance was also associated with the increased local expression
of several parameters, including transcription of a bacterial gene
encoding a presumptive araT, IL-22 production and the proportion of IL-22-producing innate lymphoid cells in the stomach
(IL-22 had an anti-Candida activity). This shows how microbiotaderived signals can elicit a host response that underlies colonization resistance to fungi.
Finally, therapeutic administration of IAld to WT mice with
either mucosal candidiasis or dextran sodium sulphate induced
colitis, restored anti-Candida resistance, promoted mucosal protection from injury and ameliorated the symptoms of colitis.
These effects were not found in mice lacking the AhR receptor,
and which were therefore insensitive to the immunostimulatory
effects of IAld.
Thus, specific microbiota interventions that would harness
the immunomodulatory potential of its constituent microbes
to boost the host’s intrinsic antifungal resistance could be
developed.
Several clinical trials have shown that probiotic strains of
bacteria or fungi can be used prophylactically to prevent fungal
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outgrowth or infection following antibiotic treatment. One such
prospective, double-blind, randomized controlled trial based on
the Pediatric intensive care unit of a hospital in India (Kumar et al. 2013) enrolled 150 critically ill children who were being treated with broad-spectrum antibiotics, and gave them either a probiotic cocktail or a placebo as part of their treatment
regime for 14 days. The probiotic cocktail contained a mixture of
bacteria and yeast species—two Lactobacillus (L. acidophilus and
L. rhamnosus), two Bifidobacterium (B. longum and B. bifidum) and
two Saccharomyces (S. boulardii and S. thermophiles) species—, in
addition to prebiotics (fructooligosaccharides) on a lactose base,
while the placebo group received only lactose. The probiotic
treatment led to significantly fewer individuals testing positive
for the presence of Candida species in rectal swabs taken 14 days
after enrolment in the study. The incidence of candiduria was
also significantly decreased in the probiotic-treated group, although there was no significant effect on the prevalence of candidemia in these children (Kumar et al. 2013).
In a separate prospective study involving 249 preterm newborns in Italy (Romeo et al. 2011), Lactobacillus probiotics (L. reuteri
ATCC55730 and L. rhamnosus ATCC53103) were also found to be
effective at reducing enteric Candida colonization and improving several other clinical parameters. Another prospective, randomized, double-blind, placebo-controlled study which focused
on 112 preterm low birth weight neonates also found that supplementation with a probiotic cocktail of Lactobacillus and Bifidobacterium species led to a significant reduction in enteric Candida levels, and a reduced incidence of invasive fungal sepsis
(Roy et al. 2014). Evidently, dietary supplementation with probiotic bacteria can be a useful clinical strategy to overcome fungal
infections in humans, but little is known of the utility of fungal probiotics alone for the same purpose. The fact that yeasts
are likely to be resistant to most antibiotics is a favourable attribute of yeast-based probiotics. Thus, unlike bacterial probiotics which may be sensitive to a given antibiotic, yeast probiotics could potentially confer beneficial effects on the host,
even during antibiotic treatment. Although, the literature describing the anti-fungal potential of fungal probiotics is sparse,
several animal trials have attested to the probiotic potential of
S. boulardii (Berg et al. 1993; Jawhara and Poulain 2007). This
yeast is the active biological component of commercially available probiotic products [UltraLevure, (France), Perenterol (Germany), Reflor (Turkey), Florastor (USA), Sacchaflor, (Denmark)].
To our knowledge, the only published clinical trial to date to examine the anti-fungal potential of S. boulardii in humans was
a prospective, randomized comparative study of very low birth
weight infants (Demirel et al. 2013). This trial showed that consumption of S. boulardii was as effective as the anti-fungal agent
nystatin, for the prevention of fungal colonization and invasive
fungal infections in these premature infants. Additional clinical
trials are needed if S. boulardii is to be adopted as a prophylactic treatment for fungal infections in humans, particularly given
that sepsis caused by S. boulardii is potentially a serious problem for immunocompromised individuals or those consuming
antibiotics (Thygesen, Glerup and Tarp 2012).
WHAT IS THE EFFECT OF C. ALBICANS
CARRIAGE ON THE GI MICROBIOTA UNDER
DYSBIOTIC CONDITIONS?
This issue was addressed by several studies from the same research group, in which the impact of the introduction of C.
albicans into a GI microbial community that was previously
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FEMS Yeast Research, 2015, Vol. 15, No. 7
Figure 1. Impact of antibiotic treatment on microbiota dynamics and on C. albicans colonization and establishment in the gut. (A) The ‘normal’ microbiota contains a diverse community of bacteria (rods and small cocci in diagram), fungi
(large white and coloured circles) and other microorganisms. Together, these
impart colonization resistance against infections by opportunistic pathogens.
(B) Antibiotic treatment eliminates subpopulations of the bacterial community.
This microbiota perturbation disturbs the colonization resistance and allows
C. albicans to establish or outgrow in this niche. In doing so, C. albicans may outcompete the other yeast species of this ecosystem. (C) Withdrawal of the antibiotic treatment allows for microbiota recovery and a reduction in the C. albicans
load. Candida albicans carriage may alter the recolonization dynamics, and could
therefore delay or prevent restoration of the pre-antibiotic treatment state. Furthermore, C. albicans may become established at low levels in this niche, which
means that relapsing infections could occur if antibiotic treatment is resumed.
disturbed by antibiotic treatment was investigated (Mason et al.
2012a; Erb Downward et al. 2013). The antibiotic of interest was
cefoperazone, a broad-spectrum antibiotic which functions by
inhibiting cell wall synthesis (DrugBank DB01329), and which is
poorly absorbed from the GI tract (Erb Downward et al. 2013). It
was administered ad libitum via the drinking bottles for 7 days
prior to the introduction of C. albicans. Data from these studies
have demonstrated, as expected, that broad-spectrum antibiotic
treatment substantially alters the structure of the GI bacterial
community. In some instances, recovery to the pre-treatment
state can be lengthy (Mason et al. 2012b; Erb Downward et al.
2013) and during the intervening period, the microbiota may be
considered as being in a state of flux. Consequently, the colonization resistance that is usually imparted by the undisturbed
microbiota towards opportunistic pathogens and transient colonizers is lowered during this recovery period. As a result, outgrowth of particular microbial species, such as C. albicans, may
ensue (Fig. 1).
The effect of C. albicans on the microbiota of antibiotictreated and untreated mice was assessed by 16S rRNA gene targeted amplicon sequencing of DNA recovered from the cecal
mucosa on days 7 and 21 after the cessation of antibiotic treatment (Erb Downward et al. 2013). A substantial loss of diversity was apparent on day 7 in mice treated with cefoperazone
alone, but this was largely restored by day 21. The introduction of
C. albicans into a disturbed microbial community further reduced its diversity, though the reduction was not statistically
significant (Erb Downward et al. 2013). Microbiota diversity was
largely but incompletely restored in animals that had received
antibiotics and C. albicans by day 21.
Candida albicans carriage influenced recovery dynamics. For
example, on day 7, in the family Ruminococcaceae the representation of the genus Oscillibacter was substantially reduced
in antibiotic-treated animals in which C. albicans was also
present. Among the Lachnospiraceae, Coprococcus and Dorea levels were reduced while an outgrowth of Roseburia and Robinsoniella species was observed. In fact, the animals inoculated
with C. albicans and treated with antibiotics had a microbiota
profile that was significantly different from those of untreated
or antibiotic-only treated animals, implying that the presence
of C. albicans influenced community reassembly. In contrast, introducing C. albicans into immunocompetent mice without an
antibiotic-induced microbiota disturbance did not significantly
alter bacterial diversity on either day 7 or 21.
Another study also investigated the associations between antibiotic treatment, GI microbiota and immune responses on C.
albicans colonization of the murine GI tract (Shankar et al. 2015).
These authors found that antibiotic treatment was the factor
that most consistently influenced C. albicans colonization. Upon
inspection of the mycobiota of the antibiotic-treated mice, it
was found that once it was introduced, C. albicans tended to displace the resident GI fungi, such as C. tropicalis. In stool samples, species of the genera Streptococcus and Parabacteroides were
associated with high levels of C. albicans, while the genera Lactobacillus and Prevotella were associated with lower C. albicans
colonization levels. Enterococcus and Veillonella were associated
with higher levels of C. albicans in the terminal ileum. The proportional abundance of each of these genera tended to be influenced by the antibiotic treatment regime chosen. Together these
studies exemplify the close relationship that exists between C.
albicans, the microbiota and their host.
By analyzing both the bacterial and fungal components of
the microbiota following defined perturbations and over time,
we broaden and deepen our understanding of the microbial population dynamics and interactions occurring in the gut ecosystem. A thorough mining of such data could potentially identify
specific bacterial populations or communities that usually keep
opportunistic fungal pathogens, such as C. albicans, in check.
While some studies of this nature have already been published
(Shankar et al. 2015; Erb Downward et al. 2013), these examples
often study C. albicans in the context of the murine gut microbiota. Equivalent data from human or humanized subjects are
lacking. Thus, before the natural colonization resistance of the
human gut microbiota towards C. albicans can be fully exploited,
we need to better understand the communities of microbes or
the specific microbial functionalities that are responsible for the
anti-C. albicans effect. Methods, models and study cohorts that
would allow the research community to address these needs are
therefore highly sought and are likely to direct future work in
this area.
PHENOTYPES AND GENES REQUIRED FOR
C. ALBICANS COMMENSALISM
Even though C. albicans’ default lifestyle is commensalism in
the GI tract, its ability to cause potent disseminated disease
means that its reputation as a pathogen often overshadows
its commensal tendencies. Many infections with C. albicans
are, however, typically opportune. Candida albicans is a polymorphic fungus, capable of occurring in a unicellular yeast
form, in one of two filamentous forms (pseudohyphae or hyphae), and also as a chlamydospore (reviewed in Whiteway
and Bachewich 2007). Particular morphotypes and the phenomenon of phenotypic switching in response to environmental triggers have been associated with commensalism and
pathogenesis (Gow et al. 2011) (Fig. 2). Thus, a precise understanding of the attributes of the various C. albicans phenotypes and their role in either commensalism or pathogenesis is
important.
In its unicellular form, C. albicans may adopt one of three distinct, non-genetically determined phenotypes named for their
colony appearance: white, opaque or gray (Tao et al. 2014). Each
Neville et al.
7
Figure 2. The C. albicans and host factors affecting C. albicans colonization levels of the murine GI tract. The laboratory mouse, which is the focal point of this figure,
has been extensively used as a mammalian host to decipher many aspects of the biology of C. albicans in the GI tract. Colonization results from the interplay between
the host and the colonizing yeast. Features that are known to influence GI colonization are listed in panels within larger boxes representing either the colonizing C.
albicans cell (top) or the host (bottom). Within each panel, arrows indicate if a given factor increases or decreases the C. albicans colonization levels. (A) A list of proteins
which regulate C. albicans colonization levels. Of the proteins listed, all but Hog1 (a mitogen-activated protein kinase) are transcription factors. (B) When compared to
the white, yeast form cells, the other C. albicans morphologies have enhanced or reduced colonization levels as indicated. (C) Microbiota composition and interventions
that modify it, such as probiotic treatment or host diet, can also influence the C. albicans GI colonization levels as indicated. (D) A functional host immune system,
genetic polymorphisms or genetic background may also impact on C. albicans levels in the GI tract as indicated. P = pathogenic infection, ? = unknown effect, - = no
effect. O/E = overexpression, KO = knockout. WT = wild-type.
of these three morphologically distinct phenotypes is stable and
can be inherited over several generations. In vitro, cells of the
different types exhibit distinguishable transcriptional profiles,
secreted aspartyl protease activities and mating competencies
(Tao et al. 2014). They also have varying virulence in in vivo models of candidiasis and cutaneous infection. White cells were
more virulent than either opaque or gray cells in a mouse model
of systemic candidiasis, while gray and opaque cells caused
more skin damage than white cells when assessed 24 hours after
being applied to the skin of a newborn mouse (Tao et al. 2014).
Gray cells were found to grow at a faster rate than either the
opaque or the white cells in an ex vivo murine tongue infection
assay, suggesting that gray cells were better adapted for nutrient
acquisition from host tissues (Tao et al. 2014).
It has been suggested that passage through the GI tract
induces a specific developmental program in C. albicans
which primes the yeast for commensalism (Pande, Chen and
Noble 2013). Consequently, its morphology, transcriptome and
metabolism may become altered, though colonizing cells usually occur in the yeast form (White et al. 2007; Pierce and
Kumamoto 2012). WT white C. albicans cells colonize the GI
tract of antibiotic-treated mice at high levels, while opaque cells
are comparatively attenuated for colonization (Pande, Chen and
Noble 2013). White cells lacking the transcription factor, Wor1,
whose levels dictate whether C. albicans is in the white or opaque
state, are rapidly cleared from the mouse gut, implying that
activation of this gene is essential for commensalism. Accordingly, overexpression mutants of WOR1 demonstrated enhanced
colonization of the murine GI tract relative to the white WT
strain (Pande, Chen and Noble 2013). The predicted alteration
of morphology during GI colonization or transit was stimulated
by the recovery of WOR1 overexpression mutants with an unexpected ‘dark’ phenotype. These cells were distinguishable from
opaque cells by having a phenotype that was stable at elevated
temperatures (>25◦ C), bearing no surface pimples, containing
prominent vacuoles, having a heterozygous mating-type locus,
not responding to mating pheromones and by being less efficient at mating (Pande, Chen and Noble 2013). The ‘dark’ cells
also had an altered transcriptome which appeared to optimize
yeast behaviour and metabolism for GI colonization. However,
the ‘dark’ phenotype was only observed with the WOR1 overexpression strain, prompting the suggestion that the host factors that are responsible for the white-to-dark transition are not
well replicated in vitro. It was hypothesized that the stable overexpression of WOR1 (which for WT cells would ostensibly be
triggered by GI transit) in the mutant strain allowed the altered
8
FEMS Yeast Research, 2015, Vol. 15, No. 7
‘GUT’ (Gastrointestinally-indUced Transition) phenotype to be
observed in the laboratory.
Thus, the phenotypes expressed by C. albicans may reflect different functional specializations and developmental programs
that have been optimized for either commensalism or pathogenesis in different niches. While these phenotypes most likely
reflect the coordinated expression of a particular panel of genes,
several studies have identified specific genes with critical roles
in either commensalism or pathogenesis.
One such gene is SFU1, which promotes C. albicans commensalism in the GI tract (Chen et al. 2011). This gene is part of
the unique tripartite iron utilization system of C. albicans and
confers resistance to toxic levels of iron in the gut by restricting iron uptake in this iron-rich environment (Chen et al. 2011).
In contrast, Sef1, a transcriptional activator of both iron uptake
and iron-utilization genes (amongst other targets), was necessary for virulence in bloodstream infections in which iron was
poorly available due to its sequestration by the host. Nevertheless, relative to the WT strain, mutants lacking either SEF1 or
SFU1 were impaired in their ability to persist in the murine gut
over a 15-day period (Chen et al. 2011). Interestingly, the GUT
cells described by Pande, Chen and Noble (2013) were also found
to downregulate expression of iron-acquisition genes, which is
consistent with their apparent optimization for GI colonization.
Thus, signalling pathways that relay signals from the host
or the environment are likely to influence gene expression in
the colonizing yeast. The mitogen-activated protein kinase Hog1
was found to be essential for colonization of the murine GI tract
by C. albicans (Prieto et al. 2014). The HOG pathway is involved
in adaptation to osmotic and oxidative stresses and has a role
in cell wall biogenesis (Prieto et al. 2014). Mutants lacking HOG1
were rapidly cleared from the gut when in competition with the
parental strain. A Hog1 conditional mutant, in which excision
of HOG1 was made possible by induction of a flippase system,
revealed that HOG1 was needed for long-term colonization of the
mouse gut. Ex vivo assays indicated that the hog1 mutant was
sensitive to bile salts and displayed poorer adhesion relative to
the WT strain (Prieto et al. 2014). Two other MAP kinases, Cek1
and Mkc1, were also tested in a competition model with a CAF2
mutant (parental strain with a reporter gene, considered as WT),
and while they initially colonized the GI tract as well as the WT,
this stable colonization was not maintained for longer than 2 to
3 weeks (Prieto et al. 2014).
The transcription factor Efg1, which is central to the regulation of the C. albicans yeast-to-hypha transition, has also been
identified as a major regulator of GI colonization in mice (Pierce
and Kumamoto 2012). The expression levels of this gene are influenced by the immune status of the host and the time course
of colonization (Pierce and Kumamoto 2012). With reference to
expression levels in vitro, transcription of EFG1 was low during
the early phases of colonization of the murine ileum and Cecum
but tended to increase with time in immunocompetent hosts
(Pierce and Kumamoto 2012). In contrast, in immunocompromised mice, EFG1 expression remained low throughout the
experimental period. Thus, C. albicans’ gene expression during
colonization is influenced by the immune status of the host. A
natural heterogeneity of EFG1 expression levels is thought to exist in the colonizing yeast population. This ‘phenotypic diversity’ may allow the C. albicans community to respond appropriately to host-imposed signals to ensure successful persistence
and niche colonization (Pierce and Kumamoto 2012).
The transcriptome of C. albicans cells in the GI tract differs
markedly from that of laboratory grown cultures (Pierce et al.
2013). Moreover, while the transcriptional profile of yeast cells
colonizing different regions of the GI tract overlap considerably, they can also be distinguished, reflecting adaptation by
the yeast to colonization of the anatomically and physiologically distinct niches of either the ileum or the Cecum (Pierce
et al. 2013). In general, according to GO terms enrichment, colonization requires expression of genes apparently involved in
pathogenesis (but which are more likely to represent cell surface restructuring for adhesion or immune evasion during colonization, given that most colonizing cells occur in the yeast
rather than hyphal forms), carbohydrate metabolism and stress
response. Comparative studies involving various EFG1 knockout
mutants revealed that Efg1 directly influenced expression of the
Als3 adhesin, hypha-regulated cell surface protein Hyr1, the secreted aspartyl proteases Sap4 to Sap6 and the Sod5 superoxide dismutase (Pierce et al. 2013). Genes involved in pathogenesis and oxidative stress responses were generally expressed at
lower levels in the efg1 mutant strain than in the WT during
colonization, while genes involved in lipid catabolism were expressed at higher levels in the mutant. This suggests that Efg1
also influences the in vivo expression of these ‘host-response’
genes, and the influence of Efg1 was more pronounced for cells
colonizing the Cecum than the ileum (Pierce et al. 2013).
An earlier study from the same group showed that Efh1,
a transcription factor paralogous to Efg1, was also highly expressed in the porcine and murine oro-GI tracts during colonization by C. albicans (White et al. 2007). A mutant strain lacking EFH1 was capable of enhanced colonization relative to the
WT strain. This echoes the results achieved with Efg1 knockout mutants above (Pierce and Kumamoto 2012). Thus, expression of EFH1 and EFG1 in vivo provides a reversible way to
limit the size of the colonizing C. albicans population (White
et al. 2007; Pierce and Kumamoto 2012) perhaps in response to
host-derived signals. Transcription factors are therefore interesting candidates for studies aiming to decipher the means by
which C. albicans persists as a commensal in its host. Nevertheless, the influence of transcription factors on GI colonization likely represents the first step in a cascade of events in
which the transcriptome of the colonizing yeast is reorientated
so that the genes encoding the true ‘effector’ molecules are expressed. The challenge therefore lies in determining not only
which genes are essential for GI tract colonization, but also in
elucidating the mechanism(s) by which these genes exert their
effect.
This was the approach taken in a comprehensive study of
77 C. albicans transcription regulator mutants (Pérez, Kumamoto
and Johnson 2013). Using mouse models, eight such factors were
identified as being required for GI colonization, systemic infection or both. Of these, three regulators (Rtg1, Rtg3, Hms1) were
involved in both colonization and infection, while two (Lys144,
a zinc-finger cluster transcription factor and Tye7, which is involved in carbohydrate metabolism) were needed for intestinal
colonization only. Considerable overlap exists in the targets of
these regulators, suggesting that colonization and systemic infection are controlled by the same network. The subset of effector genes under the influence of Rtg1 and Rtg3 was found
to be particularly upregulated in the GI tract, implying a significant role for these two regulators during colonization. These
and some of the aforementioned regulators were shown to bind
upstream of genes for amino acid permeases and allantoate
transporters, expression of which is thought to facilitate nitrogen acquisition in the gut environment (Pérez, Kumamoto and
Johnson 2013). Homozygous deletion mutants of GAL10, DFI1
and HAP41, genes that are believed to be regulated by Hms1 and
Rtg1/3, demonstrated impaired GI colonization. Expression of
Neville et al.
these genes may lead to cell surface remodelling, which therefore influences colonization.
Unravelling the genetic programs behind C. albicans commensalism and pathogenicity (Fig. 2) could potentially allow for
the development of drug-based interventions which would reduce the risk of C. albicans infections in colonized or ‘at risk’ individuals. While much progress has already been made towards
this goal, more is needed, and research into this area is likely to
continue into the future.
CANDIDA ALBICANS–HOST INTERACTIONS
AND INTERPLAY DURING COLONIZATION
AND OUTCOME DURING INFECTION
Several pattern recognition receptors (PRRs) are involved in
sensing fungal microbe-associated molecular patterns (MAMPs),
and include members of the Toll-like receptor family, the C-type
lectin receptor family, the galectin family receptors and indirectly, the Nod-like receptors (Romani 2011). These receptors respond to fungal molecules such as cell wall carbohydrates, surface proteins and fungal nucleic acids (Romani 2011). The host
can respond differently to the yeast and the filamentous forms
of C. albicans and certain cell types and receptors may specifically recognize or mount a tailored immune response to either one or the other of these fungal morphotypes (reviewed in
Romani, Bistoni and Puccetti 2002; Naglik et al. 2011). This capacity of the host to distinguish yeast forms from filamentous
C. albicans cells is critical to the host’s ability to discriminate
benign commensalism from pathogenic infections (Naglik et al.
2011). Moreover, the host can sense and respond to the increased
fungal burden that is a hallmark of candidiasis, implying that
a threshold level of C. albicans exists, and levels exceeding this
threshold distinguish pathogenesis from commensalism (Moyes
et al. 2014). What follows here is a discussion of C. albicans recognition by the host during commensalism in the GI tract specifically, with particular attention given to the PRRs and cytokines
that are involved. Disagreements on these topics in published
experimental research articles will also be described.
Of the PRRs responsible for detecting fungal ligands, the Ctype lectin receptor, Dectin-1 (also known as CLEC7A) has received considerable attention. This receptor is considered by
some as essential for responding to systemic but not GI infections with C. albicans (Vautier et al. 2012). Dectin-1 is expressed
on myeloid cells (neutrophils, macrophages, dendritic cells) that
are found in the lamina propria of the GI tract and recognizes
β-1,3 glucans, which are part of the yeast cell wall. Following
systemic infection with C. albicans, mice lacking Dectin-1 exhibited higher mortality rates, higher GI tissue colonization and a
greater C. albicans load in stool when compared to WT mice with
a functional Dectin-1 receptor (Vautier et al. 2012). The cytokine
profile of Dectin-1-deficient animals was also altered, particularly in the stomach and colon. However, there was no major
alteration of inflammatory parameters in the Dectin-1-deficient
animals following systemic C. albicans infection, but evidence
for tissue invasion and altered bile salt production was recorded
among these animals. Under conditions of direct oral GI inoculation with these Dectin-1-knockout animals, no difference in the
stool load nor the tissue colonization levels of WT nor knockout animals was recorded, even when mice were cohoused (and
therefore subjected to identical antibiotic and C. albicans dosing
regimens). This observation held true, even when two clinical C.
albicans isolates, which had been recovered from mucosal infections, were used instead of SC5314, which is suspected to poorly
9
colonize GI tissues. Candida albicans elimination from the GI microbiota was also rapid in both WT and Dectin-1-deficient mice
in the absence of antibiotic treatment. Cytokine responses were
not affected by the Dectin-1 deficiency in mouse models of direct GI inoculation, except for IL-4, which although expressed at
low levels overall (<10 pg ml−1 protein) was slightly, though significantly, more abundant in the stomachs of the mice lacking
this receptor on day 14 following the introduction of C. albicans
(Vautier et al. 2012).
The findings of Vautier et al. (2012) suggest that Dectin-1 does
not play a role in host-immune responses to C. albicans carriage
in the GI tract; however, this contradicts an earlier study (Galès
et al. 2010) in which Dectin-1 was found to play a role. In the earlier study, mice were bred with a macrophage-specific Dectin-1
deficiency. They were also treated or not with a PPAR-γ stimulatory ligand, rosiglitazone. PPAR-γ is a nuclear receptor, stimulation of which (by interleukin-13 or rosiglitazone) activates
anti-fungal responses. Following oral inoculation, greater C. albicans loads were recorded in the stomachs and ceca of mice with
the macrophage-specific Dectin-1 deficiency and which were
treated with rosiglitazone (a PPAR-γ ligand) than in the control mice. Furthermore, in the absence of rosiglitazone treatment, C. albicans levels were higher in the stomachs and ceca
of macrophage-specific Dectin-1-deficient mice than in the controls. Subsequent treatment with rosiglitazone resulted in C.
albicans clearance only in the control mice, thus emphasizing
the role of Dectin-1 in the antifungal host response towards
C. albicans. The mannose receptor on macrophages also plays
a role in the host’s antifungal response, and its expression on
macrophages was increased in response to rosiglitazone treatment, but without stimulating C. albicans elimination. This implies that the mannose receptor alone is insufficient to clear C.
albicans infections of the GI tract (Galès et al. 2010). Vautier et al.
(2012) reconcile the differences between their findings and those
of Galès et al. (2010) by referring to differences in experimental
design (presence/absence of antibiotic treatment or cohousing
of WT and mutant mice). They also suggest that microbiota differences between mouse colonies could have a contributory role
(Vautier et al. 2012).
Critically, the genetic background of the mouse line has been
shown to affect the Dectin-1-knockout phenotype (Carvalho
et al. 2012), and this may affect experimental outcomes and reproducibility across laboratories and studies. Differences in innate and adaptive immune responses occur. C57BL/6 Dectin1-deficient mice were susceptible to infection with C. albicans.
These mice harbored a higher fungal load in the stomach,
showed evidence of dissemination to the kidneys and histological analysis revealed considerable inflammation and tissue invasion, when compared to WT mice. In contrast, BALB/c Dectin1-knockout mice harbored a lower fungal load in the stomach
and showed no signs of dissemination or inflammatory cell recruitment (Carvalho et al. 2012). Furthermore, the Dectin-1 deficiency resulted in different cytokine profiles upon C. albicans
challenge in each of the different mouse lines. Proinflammatory cytokines, tumor-necrosis factor-α and interleukin-6 were
higher and lower in C57BL/6- and BALB/c- Dectin-1-knockout
mice, respectively, when compared to the WT controls. In addition, the interleukin-17 family cytokines were more highly
expressed in the BALB/c Dectin-1-deficient mice than in the
controls, and these cytokines were produced at lower levels in
the C57BL/6 Dectin-1-knockout animals than in the WT mice.
These differential responses were also extended to the Ahr/IL22 signalling pathway in these different mouse lines, with these
responses being either suppressed or activated in C57BL/6- and
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FEMS Yeast Research, 2015, Vol. 15, No. 7
BALB/c- Dectin-1-deficient mice, respectively (Carvalho et al.
2012).
Dectin-1 deficiency has also been shown to regulate the
severity of chemically induced colitis in mice (Iliev et al. 2012).
Moreover, a SNP variant of CLEC7A (a Dectin-1 homolog) was
found to be associated with a severe ulcerative colitis phenotype
in humans (Iliev et al. 2012). This receptor variant was not associated with the establishment, but rather the severity of disease.
Human genetic polymorphisms may impact the outcome of
C. albicans infections, as is well demonstrated in the example
of chronic mucocutaneous candidiasis (CMC). This disease involves persistent or recurrent infections of the mucosae of the
oral and genital tracts, the skin and nails with Candida species,
often C. albicans, and typically affects individuals with T-cell deficiencies (reviewed in Cypowyj et al. 2012). The interleukin-17
(IL-17) family is a group of six cytokines (IL-17A to IL-17F) (Gaffen
2009) which play a significant role in the host’s anti-fungal response (Gladiator and LeibundGut-Landmann 2013). Several IL17 receptors (IL-17RA to IL-17RF) are known in humans (Gaffen
2009). IL-17A and IL-17F, which are principally secreted by T cells,
play a role in the resistance to mucocutaneous infections by
pathogens (Gladiator and LeibundGut-Landmann 2013).
Impaired IL-17 immunity may increase an individual’s susceptibility to CMC, and genetic studies have identified several
IL-17 deficiencies as being responsible for CMC in humans. For
example, a homozygous, premature stop codon which abolished
IL-17RA receptor expression and the subsequent cellular responses to IL-17A and IL-17F homo- and heterodimers was responsible for CMC in a child of consanguineous parents (Puel
et al. 2011). Moreover, a dominant hypomorphic missense mutation in the IL-17F gene negatively impacted the formation of
homo- and heterodimers involving this isoform (Puel et al. 2011).
In particular, receptor binding by complexes involving IL-17F
was impaired. Variations in genes such as CARD9 and STAT1
or STAT3, which act downstream of the PRRs that detect fungal ligands, and which impact IL-17 T-cell development are also
involved in susceptibility to CMC (Cypowyj et al. 2012). Despite
these findings from human studies, it has recently been suggested that IL-17-dependent immune responses are not involved
in the regulation of GI tract colonization by C. albicans in mice
(Vautier et al. 2015). Mice lacking either the IL-17A or the IL17RA genes were inoculated with a C. albicans strain (MBY38;
tetO-UME6) in which filamentation can be readily induced by the
withdrawal of doxycycline from the system. Typically, C. albicans
filamentous forms colonize the GI tract at lower levels than the
yeast form cells, thus induction of the switch from the yeast to
the filamentous morphology in vivo was expected to result in reduced C. albicans colonization levels. Indeed, for both WT and
mutant mice, the recovery of C. albicans from stool was reduced
upon induction of filamentation. However, there was no difference in the kinetics of colonization in either the WT or knockout mice lacking either the IL-17A or the IL-17RA genes during or after doxycycline treatment, and the C. albicans load in
stool from these different treatment groups was deemed similar, implying that neither IL-17A nor IL-17RA influenced GI colonization by C. albicans (Vautier et al. 2015). Other authors have
also acknowledged that members of the IL-17 cytokine family,
including IL-17A and IL-17F, are perhaps not essential for antifungal immunity; however, these authors have shown that IL-22,
an IL-10 family cytokine that is also secreted by Th-17 cells, is required for early-stage fungal resistance in mice lacking IL-17RA
(De Luca et al. 2010).
Clearly, a complex network of immune mediators is involved
in the host response to C. albicans and the outcome of infection
is likely to be influenced by the site and stage of infection, as well
as by the immune status of the host (Romani 2011). Given that
carriage of C. albicans is typically benign in immunocompetent
hosts, host immunity clearly plays a major role in preventing
infections with this yeast.
The widespread presence in healthy humans of antibodies against serodominant C. albicans antigens including those
associated with virulence traits is indicative of an ongoing
commensal-host interplay (Mochon et al. 2010). It has been suggested that the strong humoral response of healthy individuals
towards these serodominant antigens could be one way in which
colonization and outgrowth by C. albicans is controlled in these
individuals. Nonetheless, these authors acknowledge that the
strong IgG response of this healthy cohort could also reflect their
prior exposure to C. albicans during a superficial infection, such
as vaginitis (Mochon et al. 2010).
CONCLUSIONS AND OUTLOOK
The yeast C. albicans is a common member of the human
gut mycobiota with a worldwide, though variable, distribution
(Yatsunenko et al. 2012; Angebault et al. 2013). Even so, the interactions of C. albicans with the enteric yeast community and
with the gut microbiota in general are presently quite poorly
understood. While the bacterial component of the human gut
microbiota has been studied under various healthy and disease
states, the mycobiota and its potential role in host health and
well-being has been largely overlooked. In addition, few studies
have attempted to access or assess the fungal gene catalogue of
the GI mycobiome.
Although the bacterial component of the microbiota is
known for its ability to prime the immune system and maintain homeostasis in the intestine (Rakoff-Nahoum et al. 2004),
only very recently has any progress been made towards understanding the equivalent contribution made by the viral
(Kernbauer, Ding and Cadwell 2014) or indeed the eukaryotic
communities present. Given their many surface MAMPs which
can be recognized by several different classes of host PRR, yeasts
are very likely to contribute. A better understanding of the interactions between the colonizing enteric yeasts and their hosts
could elevate the role of these yeasts from commensals to symbionts, engaged in the promotion and the establishment of host
health.
An understanding of the gene networks involved in either
commensalism or pathogenesis in C. albicans could provide candidate genes or signalling pathways that could be targeted to
prevent the transition to pathogenesis. As such, investigations
of the expression and regulation of these signalling networks
during in vivo commensalism and infection are worthwhile, and
many forward genetics screens have already yielded considerable insight into the genes regulating C. albicans commensalism
in the GI tract. The next step will be to decipher the downstream
targets of these regulators and to understand the mechanisms
by which they function.
Millennia of coevolution underscore the relationship between the human host and its commensal microbiota, and
C. albicans is thought to have been an early colonizer (though it
could also represent a more recent arrival) (Odds 2010). Although
its pathogenicity has stimulated much of the research on C. albicans to date, its commensal form is also worthy of attention,
given its potential for immunomodulation, microbiota interaction and its contribution of metabolites and biomass to the gut
environment.
Neville et al.
ACKNOWLEDGEMENTS
The authors thank Natacha Sertour and Sadri Znaidi for advice,
Martin Frank and Mariachiara DiMatteo for etymological assistance and Muriel Derrien for critical reading of the manuscript.
FUNDING
This work has been supported by grants from the Agence
Nationale de la Recherche (KANJI, ANR-08-MIE-033–01; ERANet Infect-ERA, FUNCOMPATH, ANR-14-IFEC-0004), Danone
Nutricia Research, and the French Government’s Investissement
d’Avenir program (Laboratoire d’Excellence Integrative Biology
of Emerging Infectious Diseases, ANR-10-LABX-62-IBEID; Institut de Recherche Technologique BIOASTER, ANR-10-AIRT-03) to
CdE and by grants from DIM Malinf – Région Ile-de-France to
MEB. BAN was the recipient of a post-doctoral fellowship from
Danone Nutricia Research.
Conflict of interest. None declared.
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