Molecular mechanisms behind fungal-fungal and fungal-bacterial interactions
Thijs Gruntjes Bsc.
Kamilova, F. et al. (2008). “Biocontrol strain Pseudomonas fluorescens WCS365 inhibits germination of
Fusarium oxysporum spores in tomato root exudate as well as subsequent formation of new spores.”
Environmental Microbiology 10(9): 2455-2461
Supervisor:
Examinator:
Second Reviewer:
Dr. Isabelle Benoit
Dr. ir. Ronald de Vries
Dr. Joost van den Brink
Fungal physiology
Centraal Bureau voor Schimmelculturen
Koninklijke Nederlandse Akademie van Wetenschappen
Universiteit Utrecht
1
Summary
Research on fungi has mostly focused on monocultures, however, fungi are almost always interacting
with other fungi and bacteria in natural circumstances. Many such interactions have been described, but
knowledge of the molecular mechanisms underlying these interactions remains very limited. With the
development of modern scientific techniques, more and more interactions of fungi with other microbes
are beginning to be elucidated. This review will give an overview of research focusing on the molecular
mechanisms behind these interactions, separated in two categories; molecular mechanisms underlying
fungal-fungal and fungal-bacterial interactions that require direct contact and molecular mechanisms
underlying interactions that are mediated by excretory molecules.
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Table of contents
Summary
Table of contents
1. Introduction
1.1. Fungal interactions
1.2. Aims
2. Interactions via direct contact
2.1. Role of surface proteins in direct contact
2.2. Role of surface saccharides in direct contact
2.3. Endobacteria
3. Interactions via excreted molecules
3.1. Excreted enzymes
3.2. Excreted metabolites
3.2.1. Excreted saccharides
3.2.2. Excreted acids
3.2.3. Quorum sensing molecules
3.2.4. Excreted toxins
4. Conclusions
5. References
3
2
3
4
4
5
8
8
10
11
12
12
14
14
15
16
18
19
21
1. Introduction
Fungi are important to humans in a lot of ways. So far, studies done on the characteristics of fungi have
mostly investigated monocultures, however, in non-lab circumstances, fungi are almost always
interacting with other micro-organisms, such as bacteria and other fungi.
These interactions have various implications for humans, such as the food industry, where mixed
cultures are used in the manufacturing of products such as cheese, wine and tempeh (1,2). Mycorrhizal
fungi also interact with various other microbes in the soil, which can greatly influence the effects these
fungi have on the plants (3). Numerous microbes have also been described to be involved in agonistic or
antagonistic interactions with human pathogenic and phytopathogenic fungi (4,5).
Many other interactions of fungi with other fungi or bacteria have been described, however, due to
difficulties in cultivating these organisms and limited technological possibilities, this field of research is
still in its infancy. In recent years, with the expansion of biotechnological possibilities, including the
various –omics, more and more research is focused on fungal interactions with other microbes.
1.1. Fungal interactions
Most of the research done on fungal-fungal and fungal-bacterial mixed cultures are focused on the
outcome of these interactions (1). For instance, the effects of mixed cultures on production of excreted
enzymes have been described various times. Hu et al. (2011) reported improved production of
extracellular enzymes when Aspergillus niger and Aspergillus oryzae were co-cultivated with each other
and Phanerochaete chrysosporium or Magnaporte grisea. Higher activities of β-glucosidase, αcellobiohydrolase, β-galactosidase, laccase and β-xylosidase, enzymes commonly used in biotechnology,
were measured in co-cultivation compared to the respective monocultures (6). Qian et al. (2012) also
reported increased laccase activity in co-cultures of Trametes versicolor and P. chrysosporium. A higher
potential to oxidize benzo[a]pyrene, a polycyclic aromatic hydrocarbon, was also reported for these
fungi (7). Dong et al. (2012) showed an increase of laccase and manganese peroxidase in co-cultures of
Dichomitus squalens and Ceriporiopsis subvermispora and a decrease in laccase activity in co-cultures of
D. squalens and C. subvermispora (8).
Novel production of metabolites in response to co-culturing has also been reported. For example, Cueto
et al. (2001) described a new chlorinated benzophenone antibiotic, pestalone, produced by a marine
fungus of the genus Pestalotia only in co-culture with a marine α-proteobacterium (strain CNJ-328) (9).
Induction of the production of emericellamides A and B, showing mild antibacterial activities, by the
marine fungus Emericella sp. in co-culture with Salinispora arenicola, a marine bacterium, was observed
by Oh et al. (2007) (10). Bertrand et al. (2013) compared the metabolomics of more than 600 fungal cocultivations to their respective monocultures and found consistent induction of potentially new
metabolites in the co-cultures (11).
Many reports on interspecific interactions between mycorrhizal fungi and other soil microbes have been
published (12), as this is of great importance to humans. For instance, Brulé et al. (2001) showed that
the effects of the helper bacterium Pseudomonas fluorescens on the survival of the ectomycorrhizal
fungus Laccaria bicolor can be stimulatory under favorable growth conditions for the fungus and
inhibitory under unfavorable growth conditions (13). Sbrana et al. (2002) also illustrated the different
4
effects of soil bacteria on the ectomycorrhizal fungus Tuber borchii by testing the induction of fungal
growth variations by a large amount of bacterial strains, belonging to the actinomycetes, pseudomonads
and aerobic spore-forming bacteria. 101 of these bacterial strains showed antagonistic activity towards
T. borchii, where 17 strains increased mycelial growth up to 78% in comparison to monocultures of the
fungus (14). Schrey et al. (2005) measured gene expression of the ectomycorrhizal fungus Amanita
muscaria in response to the mycorrhiza helper bacterium Streptomyces sp. and suggested that the
bacterium enhances the mycorrhiza formation between the fungus and spruce trees mostly by
promoting fungal growth (15).
Microbes can also be beneficial to plants by antagonizing phytopathogenic fungi. Effects of the common
biocontrol fungus Trichoderma harzianum are well characterized, as illustrated by a study done by
Grondona et al. (1997), analyzing the antagonistic effects of T. harzianum on the common soilborne
fungal plant pathogens Fusarium oxysporum, Rhizoctonia solani, Phoma betae, Aphanomyces cochlioides
and Acremonium cucurbitacearum (16). Pseudomonas fluorescens bacteria have also been implicated in
antagonistic interactions against common root rots such as Gaeumannomyces graminis and F.
oxysporum. Thomashaw et al. (1990) demonstrated in situ production of the antibiotic phenazine-1carboxylic acid by P. fluorescens, which can suppress take-all, a root disease in wheats caused by G.
graminis (17). Kamilova et al. (2008) reported inhibition of microconidia formation of the causative
agent of tomato foot and root rot, F. oxysporum, in the presence of P. fluorescens (18).
Some of the best studied examples of fungal interactions with other microbes are interactions in
polymicrobial infections in humans. Biofilms consisting of multiple species of bacteria and fungi often
contain Candida albicans, a fungus that can live as a commensal with humans, but can cause life
threatening disease after a morphological switch from yeast growth to hyphal growth (4). Kerr (1994)
isolated C. albicans and Pseudomonas aeruginosa from three patients and demonstrated significant
inhibition of the fungus by the bacterium (19). Kerr et al. (1999) expanded on this by showing that
pyocyanin and 1-hydroxy-phenazine are the major antifungal molecules, inhibiting Aspergillus fumigatus
and C. albicans, possibly by inhibiting the yeast to mycelium transition in the case of C. albicans (20).
Other bacteria, such as Salmonella typhimurium (21) and Streptococcus gordonii (22), have also been
described to reduce the viability of C. albicans.
These are all examples of research describing the effects of fungal-fungal and fungal-bacterial
interactions, however, the molecular mechanisms causing these effects remain largely undescribed.
1.2. Aims
With the ever developing biotechnology, it has become more feasible for researchers to focus on the
molecular mechanisms involved in fungal-fungal and fungal-bacterial interactions. Understanding the
mechanisms behind these interactions will help to better understand fungi and bacteria in general,
leading to improvements in many scientific fields, such as biotechnology and human and plant
pathology. However, so far, only a very limited amount of researches have analyzed these mechanisms
in depth.
This review gives an overview of researches that have elucidated some of the components of these
interactions on a cellular level and describes some of the common mechanisms that are found (table 1).
Due to the diversity of fungal-fungal and fungal-bacterial interactions described, I will categorize the
5
researches into two categories. The first category consists of interactions that require direct cellular
contact between partners, in which the importance of surface proteins and surface saccharides become
apparent. Also, the relatively new field of endobacteria, bacteria surviving inside a fungal host, will be
discussed. The second category constitutes of microbial interactions mediated by excretory molecules.
This will be subdivided into interactions mediated by excreted enzymes and metabolites, including
saccharides, acids, quorum sensing molecules and toxins.
Table 1: Overview of articles elucidating some of the molecular mechanisms behind fungal-fungal or fungal-bacterial
interactions. References that are not included did not describe any specific molecules.
Author(s)
Bamford et al.
(2009)
Silverman et al.
(2010)
Peters et al.
(2012)
Gaddy et al.
(2009)
Cerigini et al.
(2008)
Ref.
number
22
Type of
interaction
Surface proteins
Specific molecule
described
Bacterial SspA and SspB
23
Surface proteins
24
Surface proteins
Fungal Als3P and
bacterial SspB
Fungal Als3P
25
Surface proteins
Bacterial OmpA
26
Surface proteins
and surface
saccharides
Surface proteins
and surface
saccharides
Surface
saccharides
Surface
saccharides
Surface
saccharides
Surface
saccharides
Fungal TBF-1 and
bacterial
exopolysaccharides
Fungal lectin and fungal
glucogalactomannan
Bernardo et al.
(2004)
27
Peng et al. (2001)
28
Brand et al.
(2008)
Rainey et al.
(1991)
Bianciotto et al.
(2001)
29
Partida-Martinez
et al. (2005)
Lackner et al.
(2011)
Abubaker et al.
(2013)
37
Endobacteria
Bacterial rhizoxin
38
Endobacteria
Bacterial rhizoxin
39
Excreted enzymes
Yang et al. (2009)
40
Excreted enzymes
Fungal endochitinase,
proteinase and β-1-3glucanase
Fungal LAAO
Tseng et al.
41
Excreted enzymes
Fungal chitinase, β-1-3-
30
31
Fungal mannose
residues
Fungal O-glycans
Bacterial EPS
Bacterial EPS
6
Organisms involved in
the interaction
C. albicans and S.
gordonii
C. albicans and S.
gordonii
C. albicans and S.
aureus
C. albicans and A.
baumannii
T. borchii and
Rhizobium
V. fungicola and A.
bisporus
S. cerevisiae and P.
damnosus
C. albicans and P.
aeruginosa
A. bisporus, P. tolaasii
and P. putida
G. margarita, A.
brasilense and R.
leguminosarum
Rhizopus and
Burkholderia
R. microsporus and
Burkholderia
T. aggressivum and A.
bisporus
T. harzianum and B.
cinerea
T. harzianum and R.
(2008)
Monteiro et al.
(2010)
42
Excreted enzymes
Yang et al. (2011)
43
Excreted enzymes
glucanase, β-1-6glucanase, xylanase,
protease and LAAO
Fungal chitinase, β-1-3glucanase, β-1-6glucanase, xylanase,
protease and αmannosidase
Fungal LAAO
Castagliuolo et al.
(1999)
Buts et al. (2006)
Castagliuolo et al.
(1997)
Sawhasan et al.
(2012)
44
Excreted enzymes
Bacterial toxin B
45
46
Excreted enzymes
Excreted enzymes
Bacterial endotoxin
Bacterial toxin A
47
Excreted enzymes
Cellulases and xylanases
Hildebrandt et al.
(2006)
Deveau et al.
(2010)
Duponnois et al.
(1990)
De Weert et al.
(2004)
Murzyn et al.
(2010)
Hogan et al.
(2004)
Jarosz et al.
(2009)
Boon et al. (2008)
48
Bacterial raffinose
50
Excreted
saccharides
Excreted
saccharides
Excreted acids
52
Excreted acids
Fungal trehalose and
bacterial thiamine
Bacterial citric and malic
acids
Fungal fusaric acid
54
Excreted acids
Fungal capric acid
55
Bacterial 3OC12HSL
Xu et al. (2008)
58
Cugini et al.
(2010)
Gibson et al.
(2008)
Morales et al.
(2010)
Moree et al.
(2012)
59
60
Quorum sensing
molecules
Quorum sensing
molecules
Quorum sensing
molecules
Quorum sensing
molecules
Quorum sensing
molecules
Excreted toxins
61
Excreted toxins
Bacterial PMS
62
Excreted toxins
Bacterial PCA
49
56
57
Bacterial CSP
Bacterial BDSF
Fungal Cyr1P and
bacterial muramic acids
Fungal farnesol
Bacterial 5MPCA
7
solani
T. harzianum, R. solani,
M. phaseolina and
Fusarium spp.
T. harzianum and R.
solani
S. boulardii and C.
difficile
S. boulardii and E. coli
S. boulardii and C.
difficile
T. clypeatus,
Sordariomycetes and
Cladosporium
G. intraradice and P.
validus
L. bicolor and P.
fluorescens
H. crustuliniforme and
P. involutus
F. oxysporum and P.
fluorescens
S. boulardii and C.
albicans
C. albicans and P.
aeruginosa
C. albicans and S.
mutans
C. albicans and B.
cenocepacia
C. albicans
C. albicans and P.
aeruginosa
C. albicans and P.
aeruginosa
C. albicans and P.
aeruginosa
A. fumigatus and P.
aeruginosa
2. Interactions via direct contact
Direct contact between fungi and other micro-organisms is often very important to their interactions.
The micro-organisms recognize each other and often attach to each other, which can result in the
formation of mixed-species biofilms. Various surface proteins and saccharides have been shown to be
involved in this direct contact.
2.1. Role of surface proteins in direct contact
A quite well studied fungal-bacterial biofilm association is that of the opportunistic human pathogenic
fungus C. albicans and various bacteria, because C. albicans regularly colonizes human mucosa and
prosthetic surfaces (23) and is regularly found in the lungs of cystic fibrosis (CF) patients (4). A variety of
bacterial species has been shown to coexist with C. albicans, modulating its growth via both bacterial
and fungal surface proteins.
Bamford et al. (2009) studied interactions between Streptococcus gordonii and C. albicans, which are
often found together in human mucosa. It was found that complex interactions between S. gordonii and
C. albicans lead to enhanced biofilm formation, as measured by increased hyphal growth and increased
biomass (22). Using S. gordonii sspA/sspB knockout strains, it was shown that the S. gordonii cell wall
proteins SspA and SspB are involved in cell-cell contact with the fungus, promoting formation of mixedspecies biofilms. Silverman et al. (2010) expanded on this by investigating the role of hyphal cell-wall
glycoprotein Als3P using a C. albicans ALS3 deletion mutant strain (23). This mutant was unable to form
biofilms on salivary pellicle or on previously deposited S. gordonii, and S. gordonii was unable to attach
to hyphae produced by the ΔALS mutant, while it was shown to clearly attach to wild-type C. albicans. S.
gordonii surface proteins SspA and SspB were again shown to be involved in the attachment of these
bacteria to C. albicans hyphae, but it is likely that many more surface components of S. gordonii can
interact with C. albicans. To test only the effect of SspB, Lactococcus lactis was transformed and
confirmed by immunofluorescence to heterologously express SspB. Wild-type L. lactis was shown to
have little interactions with C. albicans wild-type, but the L. lactis expressing SspB showed significantly
more binding to the fungus. The C. albicans ΔALS mutant was also tested, and no binding to L. lactis
expressing SspB was found. It was concluded that the S. gordonii SspB directly interacts with C. albicans
Als3P, providing a mechanism for binding of S. gordonii to C. albicans hyphae (23). A similar study on the
interactions of Staphylococcus aureus and C. albicans further support the importance of C. albicans
Als3P (24). C. albicans mutant strains lacking Als3P showed severely reduced adhesion force, measured
by atomic force microscopy, between the two species compared to co-cultivations of wild-type C.
albicans and S. aureus. By transforming Saccharomyces cerevisiae to heterologously express Als3, it was
further confirmed that the C. albicans Als3P plays an essential role in this association (24).
Bacterial surface proteins have been shown to also have inhibitory effects on C. albicans, as shown in
the case of Acinetobacter baumannii (25). By using a A. baumannii ΔompA mutant, the effect of OmpA
on attachment of the bacteria to C. albicans hyphae was investigated and found to be essential for
attachment. Laser scanning confocal microscopy and TUNEL assay, testing fungal cell death due to
apoptosis, confirmed fungal apoptosis induced by A. baumannii, but the ΔompA mutant did not induce
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fungal apoptosis. It was concluded that OmpA is essential for attachment of A. baumannii to C. albicans
hyphae, and this attachment is essential for fungal cell death (25).
Recognition by surface molecules is essential in many mixed-species interactions. Lectins are nonenzymatic proteins that can bind carbohydrate structures, often acting as recognition molecules
between micro-organisms (26). An example of a fungal lectin important for recognition is the binding of
the Agaricus bisporus fruit body lectin to Verticullium fungicola cell wall glucogalactomannan, which
leads to dry bubble disease of cultivated mushrooms (27). First, the researchers showed that
germinated spores of V. fungicola clearly showed agglutination when a protein extract from A. bisporus
fruit body cell walls was added, and it was suggested that a lectin from this protein extract was able to
interact with a specific carbohydrate. Immunofluorescence then showed that glucogalactomannan was
able to bind to the A. bisporus fruit body, but not to the A. bisporus vegetative mycelium, which does
not present the lectin. Using SDS-PAGE and MALDI-TOF mass spectrometry, the A. bisporus lectin was
identified as a tetrameric glycoprotein. Various sugars were tested for binding to the lectin using the
hemagglutination inhibition assay, and purified V. fungicola glucogalactomannan was found to have the
strongest effect. The specific binding of the surface polysaccharide glucogalactomannan to the A.
bisporus fruit body surface lectin explains why the dry bubble disease only affects the fruit bodies of A.
bisporus (27).
Fungal lectins have also been showed to be involved in the selective binding to symbionts, like the Tuber
borchii fruiting body-1 (TBF-1) protein (26). This protein is a non-glycosylated polypeptide chain found
on the hyphal cell wall and is the main soluble protein in extracts of the T. borchii fruiting body.
Rhizobium bacteria are well known for their nitrogen-fixing ability and are often found in symbiosis with
fungi and plants. Using a hemagglutination inhibition assay, the binding of the TBF-1 protein of T. borchii
to an exopolysaccharide extract of various strains of Rhizobium was compared, and it was found to only
bind to exopolysaccharides from Rhizobium strains isolated from T. borchii ascoma. This highly specific
binding suggests that the protein plays an active role in selecting these bacteria (26).
A variety of surface proteins has been shown to be important in fungal mixed-species interactions by
direct contact, of which several seem to be important for recognition, although much more research is
needed. As more of these surface proteins are identified, a better understanding of the diversity and
specific functions of surface proteins will develop, which might lead to new targets for antifungal drugs.
Also, by comparing proteomics and transcriptomics between mixed and monocultures, the specific
pathways, induced by the binding of surface proteins, can be elucidated, increasing our understanding
of these interactions.
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2.2. Role of surface saccharides in direct contact
As exemplified by the glucogalactomannan of V. fungicola and the exopolysaccharides of Rhizobium
bacteria, surface saccharides are often involved in microbial mixed-species interactions.
Yeast surface saccharides have been investigated and found to be important for acidification of beers.
Peng et al. (2001) described a bacterial lectin from Pediococcus damnosus directly binding to mannose
residues on the surface of several yeasts involved in the brewing of beer (28). P. damnosus is known to
coflocculate with Saccharomyces cerevisiae, causing beer acidification. It was found that the bacterial
lectin was located on the cell surface and interacted with mannose residues in the yeast cell wall,
causing coflocculation. This effect was hardly found for Schizosaccharomyces pombe, which has cell
walls unusually rich in galactose. Using various mannose and galactose mutants of S. pombe, it was
suggested that the galactose side branches are able to shield the mannose residues on the fungal
surface, preventing coflocculation with P. damnosus (28).
Fungal surface saccharides can also be important in resistance to antagonizing bacteria, as shown in
Pseudomonas aeruginosa, which can antagonize C. albicans by hyphal killing (29). Brand et al. (2008)
reported that C. albicans mutants that displayed defective O-glycans on the cell wall were
hypersensitive to killing by P. aeruginosa bacteria, suggesting a role for O-glycans in resistance to P.
aeruginosa. Using light and scanning electron microscopy, adhesion to live C. albicans hyphae was
observed, followed by localized cell lysis when a substantial bacterial biofilm was formed on the hyphal
surface (29).
Surface polysaccharides have been shown to be involved in the attachment of bacterial cells to fungal
hyphae, which represents a first step of the mixed-species biofilm formation. Rainey et al. (1991) used
scanning and transmission electron microscopy to show fibrillar structures associated with
Pseudomonas tolaasii and Pseudomonas putida attachment to A. bisporus hyphae (30). This fibrillar
material on the cell surface stained positive for polysaccharides and was hypothesized to be involved in
rapid and secure attachment of the cells. This attachment is important for P. tolaasii to develop the
brown blotch disease and for P. putida to initiate basidiome formation of the fungus (30).
Bianciotto et al. (2001) also suggested a role for bacterial extracellular polysaccharides (EPS) in the
attachment of Azospirillium brasilense and Rhizobium leguminosarum to Gigaspora margarita
mycorrhizal structures (31). These bacteria are plant growth-promoting rhizobacteria (PGPR) and
interact with both the plants and mycorrhizal fungi. EPS mutant strains of both PGPR species were
severely inhibited in their ability to form a bacterial layer on spores and hyphae of G. margarita
compared to wild-type strains, confirming that EPS are involved in the formation of biofilms on
mycorrhizal fungal structures (31).
In microbial communities, surface saccharides have been shown to serve as targets for recognition or to
be involved in attachment of bacteria to fungal structures. As exemplified by the hypersensitivity of C.
albicans with defective O-glycans, more research on this area might yield potential new targets for
biocontrol of pathogenic fungi.
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2.3. Endobacteria
An interesting association of fungi and bacteria are the endobacteria, bacteria that live inside fungal
hyphae (32). Several fungi have recently been discovered housing various endobacteria, but the
molecular mechanisms behind their interactions remain largely undescribed. Endobacteria have been
discovered in several mycorrhizal fungi, such as bacteria belonging to the newly defined phylogroup
Cytophaga-flexibacter-bacteriodes living inside the ectomycorrhizal fungus Tuber borchii vittad. (33),
Paenibacillus spp. living inside the ectomycorrhizal fungus Laccaria bicolor S238N (34) and the bacterium
‘Candidatus Glomeribacter gigasproum’ gen. nov., sp. nov. living in the mycorrhizal fungi Gigaspora
margarita, Scutellospora persica and Scutellospora castanea (35), however, no endobacterial functions
were described for any of these interactions. Hoffman et al. (2010) showed that endobacteria are
widespread in endophytes by examining 414 isolated of endophytic fungi using light and fluorescence
microscopy. Eight families and 15 genotypes of endobacteria were found, residing in four different
classes of Ascomycota (36).
In recent years, the molecular characteristics of the association between the endobacterium
Burkholderia and the fungus Rhizopus have started to be explored. Partida-Martinez et al. (2005) used
PCR to amplify 16S rRNA from several phytopathogenic Rhizopus strains, known for causing rice seedling
blight, and described endobacteria belonging to the genus Burkholderia (37). Rhizopus fungi are known
to cause rice seedling blight using rhizoxin, an excreted macrocyclic polyketide metabolite. Using
ciprofloxacin to repress the intrafungal Burkholderia, it was found that fungal strains without symbionts
did not produce rhizoxin, which was restored upon re-introduction of the endobacterium into the
symbiont-free Rhizopus. It was concluded that the endobacteria are responsible for the production of
rhizoxin, and the authors suggests that the bacteria in turn benefit from nutrients and protection
provided by the fungus (37).
Lackner et al. (2010) expanded on this by generating Burkholderia mutants defective in either rhizoxin
production or the type III secretion system (T3SS), a system known to be involved in many bacterialeukaryotic infections (38). The Burkholderia endosymbiont is essential for formation of sporangia and
spores by the fungal host Rhizopus microsporus, and the rhizoxin-deficient mutants were, similar to
wild-type Burkholderia, able to reinfect the fungal host, that had their endosymbionts removed, and
restore their ability to sporulate. This proves that rhizoxin is not necessary for the infection process of
Burkholderia or for the initiation of fungal sporulation by the endosymbiont. The mutants defective in
the T3SS showed little infection of the fungal host, and spore formation was only sporadically found.
Using quantitative real-time PCR, the gene expression of sctC and sctU, which encode for core
components of the bacterial T3SS, was monitored in pure culture of Burkholderia and during cocultivation of the bacterium with R. microsporus. The expression of both genes was shown to be
upregulated with a threefold increase during co-cultivation, supporting the hypothesis that T3SS is
essential for fungal sporulation in this endobacterial-host interaction (38).
The field of endobacteria has only attracted very limited attention so far, but the diversity of these
bacteria in fungi and the important effects they have on the fungal host, such as toxin production or
induction of sporulation, makes it very interesting to gain a better understanding of this symbiosis.
11
3. Interactions via excreted molecules
Many microbial associations do not interact by direct contact, as they excrete intermediates into the
surroundings to affect other organisms. Many different types of excreted molecules have been
described to affect other microbes, such as enzymes and various metabolites like acids, saccharides,
quorum sensing molecules and toxins.
3.1. Excreted enzymes
Excreted enzymes in mixed cultures have been shown to be able to have both agonistic and antagonistic
effects on other microbes. Trichoderma spp. are known to be effective competitors of various other
fungi, causing green mould disease in Agaricus bisporus (39), but also commonly used as a biocontrol
agent against phytopathogenic fungi (40, 41, 42, 43). Trichoderma harzianum is a filamentous fungus
commonly found in the rhizosphere, where it causes antagonistic effects on phytopathogenic fungi such
as Rhizoctania solani (41,42,43) and Botrytis cinerea (40). Tseng et al. (2008) aimed to elucidate the total
of proteins secreted by T. harzianum in coculture with deactivated R. solani mycelium (41). To identify
proteomics, a combination of two-dimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS) was used to analyze the range of proteins secreted by T.
harzianum in the presence and absence of R. solani mycelium. Out of 35 proteins that exhibited a clear
LC-MS/MS signal, only eight could be identified. Chitinase, β-1-3-glucanase, β-1-6-glucanase, xylanase
and protease activities were found to be significantly increased in the presence of R. solani mycelium.
Chitin and β-glucan are major components of fungal cell walls, which were suggested to be the targets
of the chitinase, β-1-3-glucanase and β-1-6-glucanase. The protease may be involved in the lysing of the
fungal host by attacking structural proteins imbedded in the cell wall. A L-amino acid oxidase (LAAO) was
also identified from the extracellular proteome of T. harzianum in coculture with R. solani. This enzyme
cleaves amino acids to form ammonia, α-keto-aminocaproic acid and hydrogen peroxide. This hydrogen
peroxide is thought to be partly responsible for collapsing the fungal membrane (41).
Monteiro et al. (2010) did a secretome analysis to identify extracellular proteins secreted by T.
harzianum, growing on cell walls of R. solani, Macrophomina phaseolina and Fusarium spp (42). 60
proteins excreted by T. harzianum were analyzed by 2-DE and MALDI-TOF mass spectrometry, but only
seven were successfully identified with known functions. These also included the chitinase, β-1-3glucanase, β-1-6-glucanase, xylanase and protease that Tseng et al. (2008) reported, but an αmannosidase was also identified. Mannoses are known to be present in fungal cell walls in high
amounts, which might explain the increase in α-mannosidase activity of T. harzianum grown on fungal
cell walls. Significant differences in activity of these six enzymes were found depending on the fungal
cell wall upon which T. harzianum was grown, although they were all higher compared to the control
culture of T. harzianum grown without fungal cell wall substrate. This suggests that the levels of
expression of these enzymes excreted by T. harzianum is related to the composition of the fungal cell
wall of the phytopathogen (42).
A follow-up study done by Yang et al. (2009) used 2-DE and LC-MS/MS to describe increased activity of
the same cell wall degrading enzymes of T. harzianum in the presence of deactivated Botrytis cinerea
mycelium (40). Three enzymes were uniquely found in the presence of B. cinerea, two endochitanases
12
and LAAO. A model of fungal killing by T. harzianum was presented in which proteases and cell wall
degrading enzymes, including chitinase, β-1-3-glucanase, β-1-6-glucanase and xylanase, degrade the
fungal cell wall and anchored proteins, after which reactive oxygen species, including h2o2 resulting from
LAAO activity, collapse the fungal membrane (40). Yang et al. (2011) further characterized the T.
harzianum LAAO (Th-LAAO) by gel filtration column chromatography and subsequent MALDI-TOF mass
spectrometry in presence and absence of deactivated R. solani hyphae (43). The T. harzanium LAAO was
found to be a homodimeric protein, but in the presence of R. solani the monomeric form predominated.
Th-LAAO showed limited homology to other known LAAOs, thus it was concluded that Th-LAAO is a
novel L-amino acid oxidase. The substrate specificity of Th-LAAO was then tested, which led to the
conclusion that Th-LAAO is a L-phenylalanine oxidase. A positive correlation between inhibition of R.
solani hyphal growth and the concentration of purified Th-LAAO was found, where Th-LAAO was shown
to have a stimulatory effect on hyphal density and sporulation of T. harzianum. Preliminary results of an
experiment trying to discover whether Th-LAAO associates with the R. solani cell wall led to the
hypothesis that Th-LAAO might be able to bind to apoptosis-related cell wall proteins of R. solani,
altering their structure, followed by oxidation of these target proteins that can produce a local H2O2
concentration to induce apoptosis (43).
Abubaker et al. (2013) recently measured gene expression of three genes involved in mycoparasitism of
Trichoderma aggressivum on A. bisporus, which leads to green mould disease of A. bisporus (39). The
genes were chosen based on their roles in mycoparasitism; ech42, encoding an endochitinase, prb1,
encoding a proteinase and a gene encoding β-1-3-glucanase. It was shown that the transcription of
these three genes was upregulated when T. aggressivum was cocultivated with A. bisporus compared to
a T. aggressivum monoculture, and the increase in transcription was more pronounced when cocultured
with a sensitive strain of A. bisporus (39). This further supports the role of cell wall degrading enzyme
activities in the antagonistic interactions between Trichoderma and other fungi.
Another method of antagonism besides lysing of the target cell is inhibition of toxicity. Examples of this
are found in yeast, where secreted enzymes have been found to inhibit toxicity of bacterial endotoxins
(44,45). Saccharomyces boulardii is used as a probiotic in cases of human diarrhea caused by Clostridium
difficile, however, the direct effects of S. boulardii on C. difficile remain largely undescribed. Castagluiolo
et al (1997, 1999) used rat models to separately describe the in vitro digestion of toxin A (46) and toxin B
(44) , potent endotoxins mediating the bacterial pathogenic effect on humans, of C. difficile by a serine
protease excreted by the yeast. S. boulardii is also able to dephosphorylate endotoxins, such as LPS, on
the surface of Escherichia coli bacteria, by a protein phosphatase. This protein was purified by affinity
chromatography and shown to have a very high capacity for dephosphorylation (45).
An example of agonistic effects of excreted enzymes is given by Sawhasan et al. (2012), who showed
that fungi producing cellulases and xylanases were able to enhance the growth of Termitomyces
clypeatus, a fungus found in mounds of fungus-growing termites (47). 22 cellulase and xylanase
producing fungi, isolated from the fungus comb of termites, were selected and cocultivated with T.
clypeatus, and six of these were found to enhance the growth of T. clypeatus. Five of these fungi,
promoting the T. clypeatus growth by 85.7 – 25.7 %, shared 99% identity with Sordariomycetes
endophyte isolate 2171, based on the ITS rDNA sequences. Another isolate showed 98% similarity to
13
Cladosporium sp. CBS 280.49 and promoted T. clypeatus growth by 9.2%. The authors hypothesize that
the growth-promoting effects are explained by the excreted cellulases and xylanases aiding in
extracellular digestion of lignocellulose, making the substrates more easily available for T. clypeatus
(47).
Two different types of antagonistic interactions of fungal excreted enzymes have been described,
namely the lysing effect of cell wall degrading enzymes and LAOO of Trichoderma sp. and the inhibition
of bacterial toxins by S. boulardii. Much more research needs to be done on these subjects, as
elucidating the total mechanisms of the antagonistic interactions could be very important in biocontrol
of phytopathogenic fungi and human pathogens. In several of these studies, a mixture of excreted
proteins was isolated and identified by mass spectrometry, however, only a low amount of proteins is
identified with known functions. The characterization of new proteins, which is becoming more and
more feasible with modern technology, is essential to expand the database of known proteins, which
could give us a complete overview of the combined effect of excreted proteins in these interactions.
Research on molecular effects of excreted enzymes in mixed cultures has mostly focused on antagonistic
interactions, however, synergy of excreted enzymes of different species in the breakdown of complex
substances also deserves more attention. By describing the enzymes that different species can
contribute to the secretome of mixed-species cultures, effective combinations of microbes could be
implemented in the breakdown of plant biomass or environmental contaminants.
3.2. Excreted metabolites
Besides enzymes, many metabolites excreted by fungi and bacteria have been described to affect other
microbes in the surroundings, but the molecular mechanisms behind their effects remain largely
undescribed. Several studied have tried to elucidate these mechanisms, revealing roles of various types
of metabolites. Researches identifying and characterizing the molecules involved in the mechanisms
causing these effects will be considered here, subdivided into the involvement of saccharides, acids,
quorum sensing molecules and toxins.
3.2.1. Excreted saccharides
Excreted saccharides have been shown to be important in interactions between mycorrhizal fungi and
helper bacteria (48,49). Hildebrandt et al. (2006) investigated the stimulatory effect that Paenibacillus
validus exerts on the mycorrhizal fungus Glomus intraradice by analyzing the supernatant of a batch
culture by gas chromatography mass spectrometry (GC/MS). G. intraradice in culture could previously
not be made to sporulate without plant biomass, but cocultivation with only P. validus led to increased
fungal growth and induction of sporulation. Various bacterial sugars were identified, including raffinoselike trisaccharides, and effects of the saccharides on growth of the fungus were assessed, revealing that
raffinose significantly stimulated hyphal growth of G. intraradice (48).
Deveau et al. (2010) found another saccharide to be of importance by in the mutualism between the
ectomyccorhizal fungus Laccaria bicolor and the helper bacterium Pseudomonas fluorescens (49).
Trehalose, a non-reducing disaccharide, accumulates to high levels in L. bicolor hyphae and was found to
14
promote the growth of P. fluorescens in a concentration-dependent way, furthermore, it was
determined that trehalose acted as a potent chemo-attractant for the bacteria. Before the fungus enters
its symbiotic state with plant roots, it must import certain vitamins, like thiamine, from its environment
for efficient growth. Thiamine was shown to have a growth promoting effect on L. bicolor and P.
fluorescens was shown to be able to secrete thiamine in the same range of concentrations that
stimulates L. bicolor growth in vitro, although thiamine could not explain the total effect of P.
fluorescens on L. bicolor (49).
These investigations suggest that excreted saccharides are mainly involved in mutualistic interactions
between fungi and bacteria, however, much more research needs to be done to support this hypothesis.
Also, how the sugars exert these growth-promoting is not yet addressed. Comparative transcriptomics
between mixed and monocultures could clarify if the saccharides simply serve as a food source, or if
they are used as signal molecules to induce specific pathways involved in growth.
3.2.2. Excreted acids
Some acids, such as citric and malic acid (50) and fusaric acid (51), have also been found to be involved
in growth-promoting interactions between bacteria and fungi. Duponnois et al. (1990) described basic
mechanisms in interactions between two ectomycorrhizal fungi, Hebeloma crustuliniforme and Paxillus
involutus, with soil bacteria. The soil bacteria were found to stimulate growth of the fungi by two
distinct mechanisms; a direct effect on the fungi by stimulation of fungal growth through excretion of
the organic acids malic acid and citric acid, and an indirect effect on the fungi by metabolizing
polyphenolic substances excreted by P. involutus, which are self-inhibitory molecules that would restrict
fungal growth (50). However, the molecular mechanisms by which citric and malic stimulated fungal
growth remain undescribed.
Microbial acids also play roles in antagonistic interactions involving fungi. Fusarium oxysporum causes
tomato rot and root rot in plants, but various Pseudomonas bacteria are known to be able to control this
disease by inhibiting germination and formation of fungal spores by F. oxysporum, although the
mechanisms of this antagonism remain largely unknown (51). De Weert et al. (2004) found that, using a
P. fluorescens cheA mutant strain defective in chemotaxis, bacterial colonization of F. oxysporum hyphae
is mediated by chemotaxis (52). F. oxysporum is known to secrete fusaric acid, so the effect of this acid
as a chemo attractant was tested and found to attract the wild-type P. fluorescens in low
concentrations, but not the cheA mutant strain. Three Fusarium strains secreting different quantities of
fusaric acid were cocultivated with P. fluorescens, confirming that fusaric acid is a major chemo
attractant for the bacterium. It was suggested that the bacteria colonize the hyphae of F. oxysporum in
order to obtain nutrients secreted by the fungus (52).
The common probiotic yeast S. boulardii has been shown to inhibit the yeast to hyphae transformation
of C. albicans, which is essential for its virulence (53). To expand on this, the same laboratory focused on
characterizing the factors secreted by S. boulardii that are responsible for this inhibition (54). Using
preparative thin-layer chromatography, the extract of the S. boulardii secretome was separated into
different fractions, which were then tested for their biological activity against hyphae formation of C.
15
albicans. One fraction was shown to inhibit hyphae formation and was analyzed by GC-MS, identifying
the short chain fatty acids caproic acid, caprylic acid and capric acid. Capric acid alone was then shown
to have a inhibitory activity on C. albicans hyphae formation that was comparable to the activity
displayed by the total S. boulardii extract. Caprylic acid only showed a slight inhibitory effect and caproic
acid showed no inhibitory effect. The effect of S. boulardii extract and capric acid on gene expression of
C. albicans was also investigated and it was found that capric acid reduced the expression of HWP1,
encoding a hyphal cell wall protein important in C. albicans virulence, eight times more than S. boulardii
extract (54). This indicates that other compounds in the S. boulardii extract also modulate the effect on
the fungus, so, to understand the antagonistic effects of the biocontrol agent S. boulardii on C. albicans,
the activities and interactions of these other compounds also need to be elucidated.
3.2.3. Quorum sensing molecules
In the well-studied interactions between the pathogen C. albicans and various bacteria, bacterial
quorum sensing molecules have been shown to be of importance in inhibiting the fungal filamentation,
resulting in dominance of the non-virulent yeast morphology of C. albicans (55,56,57). Hogan et al.
(2004) showed that C. albicans filamentation is inhibited by P. aeruginosa both in liquid and on solid
media, although no effect on growth rate was found (55). Testing 14 P. aeruginosa mutant strains
against the fungus revealed that the lasR and lasL mutants, impaired in their ability to produce the 3oxo-C12 homoserine lactone (3OC12HSL) quorum-sensing signal, hardly showed the inhibition of
filamentation displayed by wild-type P. aeruginosa. A vector designed to constitutively express the lasL
gene restored the inhibition of filamentation in the lasL mutant strain. Purified 3OC12HSL was also
shown to inhibit C. albicans filamentation. Using real-time reverse transcription PCR (RT RT-PCR) it was
found that fungal expression of filament-specific genes HWP1, ECE1 and SAP5 decreased by at least 10fold in cultures with 3OC12HSL compared to control C. albicans gene expression. Yeast-associated genes
RBE1, YWP1 and RHD1 were increased in these cultures. 3OC12HSL did not only inhibit formation of
hyphae by C. albicans, but it was also found to be able to revert fungal filaments back to growth as
yeast-form cells (55).
Another quorum sensing molecule able to inhibit the morphological switch of yeast growth to hyphal
growth in C. albicans is the Streptococcus mutans competence-stimulating peptide (CSP), as described
by Jarosz et al. (2009) (56). This was shown by a strong inhibition of germ tube formation that was found
when a four hour old medium of S. mutans was added to a C. albicans culture. CSP was already known
to be produced only in the early exponential phase of bacterial growth, so it was suggested that this
peptide was partly responsible for the inhibition of germ tube formation. Furthermore, a synthetic CSP
was found to inhibit germ tube formation in a concentration-dependent manner and a S. mutans
mutant, impaired in its ability to produce CSP, showed reduced inhibition of germ tube formation. By
adding synthetic CSP, this inhibition could be restored to wild-type levels. CSP, similar to 3OC12HSL, was
also found to possibly stimulate the hyphae-to-yeast transition (56).
In another study of the inhibitory effect on C. albicans, Boon et al. (2008) identified a structural
homologue to the Xanthomonas campestris pv. Campestris diffusible signal factor (DSF), which is a
quorum sensing molecule, in Burkholderia cenocepacia and designated it as BDSF (57). Using NMR to
elucidate its structure, BDSF was found to be a cis-2-dodecenoic acid. The inhibitory effect of this
16
molecule was tested by adding it to fresh C. albicans fungal yeast cells and it was shown to cause a
significant reduction of germ tube germination and elongation. In contrast to P. aeruginosa 3OC12HSL
and S. mutans CSP, BDSF was found, at a high concentration of 100μM, to also have a detrimental effect
on C. albicans yeast growth (57).
A recent report by Xu et al. (2008) suggests Cyr1P, an adenylyl cyclase, as an intracellular target of
bacterial signal molecules in C. albicans (58). This study attempted to identify the substances in human
serum responsible for induction of C. albicans hyphal growth using chromatographic fractionations of
human plasma, which were analyzed by NMR. The results identified muramic acid (Mur), alanine (Ala)
and isoglutamine (iGln). A highly conserved subunit of bacterial peptidoglycan is MurNAc-L-Ala-D-iGln,
so this was suggested to be the substance responsible for the induction of hyphal growth in C. albicans.
The authors refer to these molecules as muramyl dipeptides (MDP) and both synthesized and isolated
them from E. coli and S. aureus and showed that they are potent inducers of hyphal growth. MDP was
found to activate Cyr1P, which triggers PKA, initiating a pathway in C. albicans that induces expression of
various hypha-specific genes, by binding directly to the leucine rich repeat domain of C. albicans Cyr1P
(58).
Besides the bacterial effect of quorum sensing molecules on C. albicans, Cugini et al. (2010) described
the effect of C. albicans on bacterial quorum sensing in P. aeruginosa mutant defective in quorum
sensing (59). P. aeruginosa lasR mutants, which lack the master quorum sensing regulator but are
commonly found in lungs of infected patients, regain the potential to produce phenazines, which are
regulated by quorum sensing, in dual-species colony biofilms with C. albicans. When added to the
medium, farnesol, a C. albicans auto-regulatory molecule, was able to stimulate production of P.
aeruginosa pyocyanin, which is a phenazine whose production is regulated by quorum sensing and
which is greatly reduced in cultures of only P. aeruginosa lasR mutants. The production of Pseudomonas
quinolone signal (PQS), which controls the expression of phenazine related genes, was found to be
increased in lasR mutant strains when farnesol was added to the LB agar. Using mutants defective in
several steps in PQS production, farnesol was shown to increase N-butyryl-homoserine lactone (C4HSL)
production, which is sufficient for PQS production in lasR mutant P. aeruginosa strains. Farnesol was
reported to lead to oxidative stress in various organisms, so it was hypothesized that this oxidative
stress leads to increased production of C4HSL. Using hydrogen peroxide to investigate the effects of
oxidative stress on the lasR mutant strain, it was shown that farnesol-induced oxidative stress is
responsible for the expression of the LasR-controlled quorum sensing pathway and subsequent
production of phenazines (59).
It seems that quorum sensing is very important in the interspecies interactions between C. albicans and
various bacteria present in human polymicrobial infections. Understanding these interactions in detail
will be clinically relevant, as possible targets for combatting and preventing candidiasis could be
revealed this way. However, much more detailed analysis of the mechanisms behind the inhibition of
hyphal formation, such as better characterization of intracellular targets responsible for this inhibition in
C. albicans, needs to be done in order to obtain a sufficient understanding of these interactions.
17
3.2.4. Excreted toxins
Toxins are important in antagonistic microbial interactions, and they are often excreted in competitive
interactions. A well-studied example of this is the interaction between P. aeruginosa and various fungi,
which is often found in polymicrobial communities in the lungs of cystic fibrosis patients (60). In recent
years, several studies have reported an interesting phenomenon (60,61,62), where phenazine
metabolites of P. aeruginosa are modified intrafungally, leading to enhanced toxicities for the fungi.
Gibson et al. (2009) cocultured P. aeruginosa and C. albicans on solid
medium, which resulted in formation of a red pigment inside the fungus,
leading to decreased fungal viability (60). To identify genes involved in this
red pigment formation, a P. aeruginosa strain PA14 Tn5 mutant library,
consisting of around 9000 random insertion mutants, was screened. Two
mutants were found to cause increased pigment formation, which were
shown to have insertions in phzF1 and phzS, genes both encoding enzymes
involved in the biosynthesis of pyocyanin (PYO), a known bacterial
phenazine (fig. 1A). Phenazine-1-carboxylate (PCA) is methylated by PhzM
and subsequently reduced by PhzS to form PYO (fig. 1B). A P. aeruginosa
phzM mutant, unable to methylate PCA, did not induce pigmentation in
co-culture with C. albicans, however, a P. aeruginosa phzS mutant, unable
to reduce the 1-carboxylate group to an alcohol, was able to cause
increased red pigmentation in C. albicans compared to the wild-type
strain. The P. aeruginosa ΔphzA1-G1 ΔphzA2-G2 mutant, defective in the
main phenazine biosynthetic genes, also did not give rise to any red
pigmentation. Because the red pigmentation required functional C
phzABCDEFG and phzM genes, but not phzS, the authors hypothesized
PMS
that the red pigment is derived from the PhzM product, which is proposed
to be 5-methyl-phenazinium-1-carboxylate (5MPCA) (fig. 1B). Further
investigations towards the red pigment showed that the pigment could Fig. 1: A) P. aeruginosa phenazine
be reversibly oxidized and reduced. By assessing the survival of C. biosynthetic genes. phzA1-G1 is
albicans in co-culture with the wild-type P. aeruginosa, the phzM mutant present twice in the genome and
and the phzS mutant strains, it was suggested that the phzM gene, which encodes enzymes necessary for PCA
production. B) phzM and phzS are
leads to the production of 5MPCA, is required for the majority of the
necessary for formation of
killing of C. albicans. This claim was further supported by red pyocyanin from PCA (60). C)
pigmentation observed in C. albicans after addition of a synthezised Structure of phenazine
PhzM product without P. aeruginosa. By using fluorescent microscopy, it methosulphate (61).
was found that the red pigment accumulated exclusively inside fungal
cells. Due to difficulties in releasing the red pigment from fungal cells, the
identity of the fungal pigment was not discovered, but it was found to be much larger than any known P.
aeruginosa phenazine. This lead to the hypothesis that the PhzM product is modified within the fungus
(60). The same group later found that phenazine methosulphate (PMS) (fig. 1C), which is commercially
available, could serve as a very suitable surrogate for Pseudomonal 5MPCA (61). C. albicans was grown
18
on agar containing PMS and a red coloration and killing of fungal cells was found. PMS was also found to
be able to be reversibly reduced and oxidized, like 5MPCA. Using mass spectrometry, it was determined
that amino-containing compounds can covalently bind to PMS and 5MPCA. This was proven in vivo by
protein precipitation, which showed that the red precipitation was associated with the pellet. The redox
potential of the phenazine was not affected by the modification inside the fungus. In accordance with
known activity of phenazines to produce reactive oxygen species (ROS), it was found, using flow
cytometry, that PMS or their derivates are able to generate ROS in vivo, which contributed to efficient
fungal killing (61).
Aspergillus fumigatus is also often found in lungs of cystic fibrosis patients in mixed communities with P.
aeruginosa, where the bacterium inhibits filamentation and biofilm formation of the fungus (62). Moree
et al. (2010) analyzed metabolites in this association by MALDI-TOF imaging mass spectrometry, which is
capable of simultaneously determining the spatial and temporal distribution of a large amount of
secreted metabolites. This technique allowed for detailed analysis of hundreds of metabolites, amongst
which bacterial phenazines were again found to be modified by the fungus. A. fumigatus converted PCA
into 1-hydroxyphenazine (1-HP), 1-methoxyphenazine (1-MP) and phenazine-1-sulfate. 1-HP was further
modified by the fungus to form 1-MP and phenazine-1-sulphate. Both 1-HP and 1-MP showed increased
toxicity towards the fungus compared to PCA, but phenazine-1-sulphate showed no antifungal activity.
Because sulfonation is a known process used by various fungi to solubilize and detoxify toxins, it is
hypothesized that 1-HP, 1-MP and phenazine-1-sulphate are intermediates in the fungal process of
detoxifying pseudomonal PCA (62).
The roles of pseudomonal phenazines are among the best studied bacterial-fungal interactions, due to
their implications in human pathology. A full understanding of these mechanisms will be very helpful in
the treatment of fungal infections in patients. Also, with the rising problem of antibiotic resistance of
pathogens, new antifungal compounds could be very helpful to deal with this problem. The mechanisms
of bacterial metabolites being converted into other molecules inside the fungus represents an
interesting type of interactions, and it would be very interesting to analyze how widespread these type
of interactions are between bacteria and fungi, and if the same type of interactions can also be found in
fungal-fungal interactions. Much more research on microbial interactions needs to be done to answer
these questions.
4. Conclusions
Many different fungal-fungal and bacterial fungal interactions have been described, however, only in a
few cases have the molecular mechanisms behind the interactions been addressed. Based on these
studies, some parallels can be drawn between the diverse interactions. Surface proteins and saccharides
seem mostly involved in the recognition and attachment of microbial partners and excreted enzymes, in
antagonistic interactions, are often involved in the breakdown of the fungal cell wall. The various
saccharides, acids, quorum sensing molecules and toxins involved in mixed-species interactions have
very different effects on the microbial partner, by very different mechanisms.
Overall, much more research is needed to elucidate the complexity of fungal-fungal and fungal-bacterial
interactions. Many experiments analyzed here only focus on one of the components of the interactions,
19
however, these interactions are often regulated by many different types of molecules. Also, often a
combination of direct cell-cell contact and excreted molecules mediate the interactions. Research on
fungal mixed cultures has to be expanded to include a larger diversity of organisms and a more detailed
analysis of the molecular mechanisms behind the interactions. Already now, with the very limited
amount of investigations presented, various new molecules and targets for either inhibition or
stimulation of certain fungi have been described. With the ever increasing development in
biotechnology, such as high-throughput proteomic, transcriptomic, metabolomics and metagenomic
methodologies and data analysis techniques, researchers will be more and more able to characterize
these highly complex and diverse interactions, which might lead to many beneficial results, such as the
description of new antibiotic agents, new ways to stimulate mycorrhizal fungi and increased production
of biotechnologically relevant enzymes and metabolites.
20
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