Medical Mycology, 2017, 55, 4–9

doi: 10.1093/mmy/myw076
Advance Access Publication Date: 8 September 2016
Review Article

Review Article

30 years of battling the cell wall

JP Latgé

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Unité des Aspergillus, Institut Pasteur, Paris
To whom correspondence should be addressed: JP Latgé, Unité des Aspergillus, Institut Pasteur, 25 rue Dr Roux, 75015
Paris, France. Tel: +33 1 40613518; Fax: +33 1 40613519; E-mail: jean-paul.latge@pasteur.fr
Received 27 May 2016; Revised 27 July 2016; Accepted 28 July 2016

Abstract
In Aspergillus fumigatus, like in other pathogenic fungi, the cell wall is essential for
fungal growth as well as for resisting environmental stresses such as phagocytic killing.
Most of the chemical analyses undertaken on the cell wall of A. fumigatus are focused
on the mycelial cell wall because it is the vegetative stage of the fungus. However, the
cell walls of the mycelium and conidium (which is the infective propagule) are different
especially at the level of the surface layer, which plays a significant role in the interaction
between A. fumigatus conidia and phagocytic cells of the immune system. In spite of the
essential function of the cell wall in fungal life, progresses have been extremely slow in
the understanding of biosynthesis as well in the identification of the key host responses
against the cell wall components. A major difficulty is the fact that the composition and
structural organization of the cell wall is not immutably set and is constantly reshuffled
depending on the environmental conditions.
Key words: Aspergillus, cell wall, polysaccharides, Entomophthorales, immune responses.

Introduction mopathogenic zygomycetes. Some of these species have

1–3 a very interesting biological cycle: when they enter
Writing another review on cell wall! What a pain . . . .
in the insect hemocoel, they form and grow as cell
Then I remembered the title of my talk suggested by
wall-less protoplastic morphotypes (Fig. 1).4,5 Because
W. Steinbach (Duke University Medical Center, Durham,
of this growth behavior and the lack of a cell wall,
North Carolina, USA), and I decided to deliver a short edi-
the fungal pathogen is not recognized as a nonself by
torial of the same title, which may look like a biopic of my
the host. Before the discovery of Dectin-1 or GNBP3
entire life devoted to the fungal cell wall. For this and to
proteins which are the β1,3 glucan receptors in mam-
separate the different stages of my life I have slightly paro-
mals and flies, I and A. Beauvais showed that it was
died the words of a song called “I know” recorded by the
the absence of β1,3 glucan and chitin on the surface
famous french movie actor Jean Gabin in 1974.
of the protoplast which was responsible for the lack of
host immune response against the pathogen. It was also
“When I was about 25, I knew everything; Oh
how J.P. Latgé and A. Beauvais met and became the
yeah, cell wall! I knew it thoroughly.”
Pierre and Marie Curie of the fungal cell wall of As-
My first encounter with the cell wall was when I pergillus fumigatus (of course with a much more modest
was working on entomophthorales, which are ento- outcome!).

4 
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Latgé
(A)
(B)
Figure 1. Morphotypes of the entomopathogenic Entomophthorales Entomophaga aulicae. A,B: Hyphal bodies; C,D: Protoplasts; E: Lack of recognition
of non-self by insect hemocytes; F: granuloma formed by the same hemocytes in response to β1,3 glucans (Beauvais and Latgé, 1988, 1989).
“Halfway through my life, I learned new things
again; What I’ve learned takes up only five words:
Cell wall is not easy.”
To understand cell wall organization, the use of method-
ologies and equipment adapted for the analysis of carbo-
hydrate and glycans is required. These methods are very
time consuming and require the chemical and enzymatic
solubilization of an insoluble armor without affecting the
inter polysaccharide linkages. Three years were necessary
to establish the structure and the specificity of the A. fumi-
gatus cell wall6 : it is composed of a fibrillar skeleton made
of a β1,3 branched glucan bound to chitin, galactomannan,
and β1,3/1,4 glucan, embedded in an amorphous alkali sol-
uble cement mainly composed of α1,3 glucan and galactose
polymers (galactomannan and galactosaminogalactan). In
contrast to yeast, cell wall proteins are only in transit in the
cell wall without playing a structural role.
Following the biochemistry era, came the molecular biol-
ogy era. It did amplify the number of publications in the area
but enlighten very little our understanding of the complex-
ity of the biosynthetic events in the cell wall construction.
As an example, the molecular analysis of the polysaccharide
synthases will be discussed below. First, sequence analysis
has led us to the in silico identification of three α1,3 glu-
can synthases (AGS). Successive deletion of the three AGS
genes lead to a fungus exempt of α1,3 glucan (40% of
the mycelial cell wall) which does not show any growth
5
H (E)
(C)
(D) (F)
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phenotype in vitro.7 However, even though the genes cod-
ing for the α1,3 glucan synthases have been identified, their
function and even their substrate remain to date unknown.
Eight chitin synthase genes were identified and clustered in
two families based on the sequence of the encoding genes.8
However, the understanding of the function of chitin syn-
thases is not helped by this clustering into two families. In
particular, the reasons for the lack of correlation between
the chitin synthase activity in vitro and the chitin content
of the cell wall that is the end product of this enzymatic ac-
tivity remain unknown. Each gene inside each family seems
also to have a different function that is not correlated to
its belonging to one family, and enzymes of family 1 and 2
have to work together to synthesize the chitin of the A. fu-
migatus cell wall. Moreover, the importance of a synthase
family is not associated to the number of genes per family.
For example, β1,3 glucan synthesis is essential in A. fumi-
gatus but in contrast to chitin synthesis depends on a single
FKS1 gene.9
These results show that fibrillar polysaccharides (chitin
and β1,3 glucan) are essential in the cell wall construc-
tion and are good antifungal targets, whereas amor-
phous polysaccharides, even though they are present in
high amount like α1,3 glucans, are not essential. How-
ever, the enzymatic activity of these essential synthases
has not been and will not be understood in vitro in the
near future because their multiple transmembrane domains
6 Medical Mycology, 2017, Vol. 55, No. 1
prohibit the production of recombinant proteins. More-
over, BLAST comparisons can identify genes in yeast and
filamentous fungi which are very homologous but end up
making a different function. This is the case of the man-
nosyltransferases of S. cerevisiae, which are responsible for
the biosynthesis of the yeast mannan, while the ortholo-
gous genes in A. fumigatus are not involved in the synthesis
of the cell wall mannans despite clear mannosyltransferase
activity.10
“I who am in the autumn of my life, You can
forget so many evenings of disappointments, But
never a morning of discovery in the lab.”
This was the case when we discovered accidently that a gly-
cosyl phosphatidyl inositol anchored protein was involved
in the remodeling of the cell wall matrix. This new enzyme
was called Gelp (glucan elongase) because it elongates the
β1,3 glucans and used an unusual long oligosaccharide of
more than 10 glucose units.11 This discovery questions the
glucan synthesis in the fungal cell: in vitro the glucan syn-
thase activity produces linear β1,3 glucans of > 1000 glu-
cose units in minutes, whereas the discovery of this transg-
lycosidase suggest that short stretches of glucans could be
produced by Fks1p and then subsequently elongated by the
Gelp proteins. However, until now nobody has shown that
Gelp and Fksp proteins work together in the same complex.
Interestingly, Gelp was not only new for science, but enzy-
matic activity was essential for A. fumigatus.12 Based on this
discovery, it was anticipated that GPI-anchored proteins
which would be common to all the fungal species should
have a role in the remodeling of the cell wall polysaccha-
rides. Four families of GPI anchored proteins are common
to all fungal species: ECM33, DFG, CRH, and GEL.13–15
If ECM33 and CRH does not seem to play any role in
the remodeling of the cell wall polysaccharides in A. fumi-
gatus, the DFG family is associated to cell wall remodeling
since dfg mutants show a defect in the covalent binding of
galactomannan to β1,3 glucans. It is clear that β1,3 glucans
is the central core of the A.fumigatus cell wall to which are
anchored different molecules such as the β1,3, β1,4 glucans
(under the control of TFT116 ) chitin and galactomannan.
These results suggest that major transglycosidases respon-
sible for the 3D construction of the cell wall and especially
the ones responsible for the branching of the β1,3 glucans
and the cross-linking with the other cell wall polysaccha-
rides are still missing. Moreover, the interaction between
hydrolases (endo β 1,3 glucanases)17 supposed to plasticize
the cell wall and synthases and transglycosidases supposed
to remodel β1,3 glucan synthesis is not well understood and
should be better investigated.
12
10
8
6 Biochemistry
Immunology
4
2
0
years Impact factor budget
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Figure 2. Graphical representation of how Latgé sees the methodologies
used to study cell wall and the consequences of the selection of the
discipline for scientists in the lab: number of years needed to publish an
article (years); impact of the journals in the discipline selected (impact
factor); budget to spend based on the selection of the discipline. The
molecular biology is not represented and would be halfway between
the 2 extremes (Biochemistry and immunology).
“The clock has stricken 60, Now I KNOW, I KNOW
THAT I should have worked on fungal
immunology earlier.”
Besides the interest in learning how aspergillosis establishes
in the human lung, embarking on a fungal immunology
study has several advantages and disadvantages explained
in Figure 2. Many of the cell wall components are recog-
nised as PAMPs and play an active role during infection.18
This role can be positive for the host, some polysaccha-
rides such as α and β1,3 glucans stimulating the defense
reaction and especially a Th1/Th17 or Treg response.18
Conversely, galactose containing polysaccharides can be
immunosuppressive and induces a Th2 type response that
favors fungal virulence. If the immune function of the galac-
tomannan has not been thoroughly investigated, the role of
galactosaminogalactan as a virulence factor because of its
immunosuppressive function, has been well documented:
(i) galactosaminogalactan is a potent adhesin which helps
the fungus to adhere to lung cells19,20 ; (ii) it is responsi-
ble for an inhibition of the neutrophil recruitment and in-
duces neutrophil apoptosis via NK cells21 ; (iii) it stimulates
the production of IL1Ra which dampens completely the
IL1 pathway, a result that has shown the essential role
of the IL1 pathway to counteract the establishment of
aspergillosis.22
PRRs recognizing cell wall PAMPs remain poorly
known. Besides Dectin-1, which shows an exquisite speci-
ficity in the recognition of the β1,3 glucans23 the other
receptors are either unknown or show a very low specificity
in terms of recognition. This may also indicate that circulat-
ing molecules such as antibodies, complement molecules or
collectins may play an important role which should be re-
visited since it has been poorly analysed to date. Indeed,
Latgé
Table 1. Major components of the cell wall and their role in A. fumigatus virulence.
Favouring the growth of the fungus in the host
r Rodlets
r Melanin
r Galactomannan
r Galectosaminogalactan
recognition of IgG opsonized chitin via the Fcγ RII and
uptake induced via the Syk/PI3K-pathway, results in iso-
lated induction of IL-1Ra. In the presence of other Syk-
independent PRR ligands, IgG-opsonized chitin induces IL-
1β in a synergistic manner.24 These results explain the dual
pro- and anti-inflammatory responses noted for this cell
wall polysaccharide in the literature.25
Interestingly, the immune potential of surface molecules
of the vegetative (mycelium) and infective (conidium) mor-
photypes is different. The rodlet hydrophobin RodA present
on the surface of the conidium immunosilences this mor-
photype because of a dual function: it is not recognized by
the immune system and prevents the cell wall PAMPs to be
exposed to induce an immediate innate immune response.26
The second surface conidial molecule that is favoring fungal
virulence is the melanin. Even though melanin is not induc-
ing a classical cytokine/ or chemokine response as well as
a Th response, it has a very strong anti-phagocytic func-
tion: (i) it blocks the acidification of the phagosome27 ; (ii)
it inhibits the NADPH oxidase that is responsible for the
production of antifungal ROS28 ; (iii) it blocks a specialized
autophagy pathway termed LC3 associated phagocytosis
which is essential in the intracellular killing of conidia by the
phagocyte.28 These studies show that cell wall components
other than polysaccharides have an immune function and
that the immune response is compound-specific (Table 1).
However, the immune function of the cell wall associated
proteins have been poorly analysed to date in A. fumigatus.
“All the time when I was young, I wanted to say I
KNOW. However, the more I searched, the less I
knew then. . . ”
One thing that I know is that the cell wall is an essential
organelle for the fungal cell. One of the difficulties to study
the cell wall is due to its continuous change and reshuf-
fling in composition and organization when its environment
changes and especially in a stress situation consecutive to
a gene deletion.29,30 If increased chitin synthesis is a well-
known compensatory reaction in response to changes of
the environment, many of these compensatory reactions are
unknown to date. For example, the composition of the cell
wall during the growth in its host has not been investigated
properly to date, whereas it would be essential to under-
7
Activating the immune response of the host
r Chitin
r β1,3glucans
r α1,3 glucans
r Proteins?
stand the efficacy of the antifungal drugs targeting cell wall
molecules. The existence of compensatory reactions also
questions some conclusions coming from the deletion of a
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cell wall gene: the phenotype of the mutant may not be
directly associated to the deletion of the gene targeted but
may be a secondary effect of the deletion that has nothing to
do with the function of the deleted gene. It has been indeed
known for many years that mutations of genes coding for
nuclear and mitochondrial proteins are associated with cell
wall modifications. It is then difficult to know if a cell wall
phenotype results from a deletion of a gene truly involved in
cell wall biosynthesis or if it is a secondary manifestation of
a gene essential for morphogenesis. This new concept of the
cell wall being a living organelle, which was not considered
few years ago, changes the way of how cell wall biosynthe-
sis must be grasped. Omic global analysis should be useful
to identify the rewiring of pathways that will become es-
sential to compensate for the deletion of a putative essen-
tial gene. Another type of compensatory reactions has been
revealed by the analysis of the immune response toward
cell wall polysaccharides. They concern the relocalization
of the different cell wall components. For example melanin
mutants induce a cytokine response and activate DC even-
though the conidia are covered with rodlets.31 This is due
to the presence on the surface of the rodlet layer of this
mutant to a layer of (glycosyl) proteins, which is respon-
sible for the induction of this immune response. Delocal-
ization of cell wall components has also been seen on the
conidia of the α1,3 glucan synthase and rodlet mutants3,32
and on mycelium treated by inhibitors of β1,3 glucan
synthase.33
What is the future in cell wall polysaccharide research?
On the biosynthetic side, the branching of β1,3 glucans
and the glucan-chitin cross-linking are certainly the events
to elucidate. On the host response side, it would be also
essential to identify all PRRs recognizing the cell wall com-
ponents. Based on the work performed with Dectin-1, it is
clear that the identification of specific receptors is a “must”
to be able to identify the pathways downstream to these
receptors. Another fallow territory is the understanding
of how MHCII present processed fragments of cell wall
polysaccharides to T cells34 ; little is known about the nature
of T cell recognition of the polysaccharide-MHCII complex
or the phenotype of the resulting activated cells.35
8 Medical Mycology, 2017, Vol. 55, No. 1
Figure 3. Biochemical knowledge of polysaccharide synthesis 30 years ago (A), and in 2016 (in bold and italics; B) with question marks for problems
to be solved in the next 30 years.
However, Now I KNOW, I KNOW THAT YOU
NEVER KNOW! And as an example, Figure 3 will sug-
gest that at least another 30 years of battling with the cell
wall will be necessary to advance in the understanding of
the life of this fascinating organelle.
Acknowledgments
This paper is dedicated to the members (present and past) of the
Aspergillus unit.
Declaration of interest
The authors report no conflicts of interest. The author alone is
responsible for the content and the writing of the paper.
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