Journal of Medical Microbiology (2014), 63, 15–27
DOI 10.1099/jmm.0.064444-0
Role of carbonic anhydrases in pathogenic microorganisms: a focus on Aspergillus fumigatus
Review
Jaqueline Moisés Tobal and Márcia Eliana da Silva Ferreira Balieiro
Correspondence
Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, São Paulo, Brazil
Márcia Eliana da Silva Ferreira
Balieiro
[email protected]
Received 20 June 2013
Accepted 20 October 2013
Aspergillus fumigatus is a ubiquitous saprophytic fungus responsible for organic material
decomposition, and plays an important role in recycling environmental carbon and nitrogen.
Besides its important role in the environment, this fungus has been reported as one of the most
important fungal pathogens in immunocompromised patients. Due to changes in CO2
concentration that some pathogens face during the infection process, studies have been
undertaken to understand the pathogenic roles of carbonic anhydrases (CAs), well-known CO2
hydration catalytic enzymes. As a basis for a discussion of the possible roles of CAs in A.
fumigatus pathogenicity, this review describes the main characteristics of the A. fumigatus
infection and the challenges for its treatment. In addition, it gathers findings from studies with CA
inhibitor drugs as anti-infective agents in different pathogens.
Introduction
Aspergillus fumigatus
It is a given that the fungal community contributes to ecosystem dynamics. Aspergillus fumigatus, for instance, is a
widespread fungus that plays its role as a saprophyte very
well. Its natural ecological niche is the soil wherein it
survives and grows on organic debris, decomposing the
organic matter and returning important inorganic nutrients such as carbon and nitrogen to the environment
(Latgé, 1999; Mullins et al., 1976).
A. fumigatus sporulates abundantly, releasing thousands of
small airborne conidia (2–3 mm in size) that can survive a
wide range of environmental conditions, eventually reaching the human bronchioles or alveoli. In immunocompetent individuals, inhalation of A. fumigatus conidia will
rarely cause severe consequences for their health, since the
innate immune system generally eliminates the conidia
efficiently. On the other hand, when inhaled by immunocompromised patients, A. fumigatus becomes an opportunistic pathogen and a serious public health problem.
With A. fumigatus, the virulence is multifactorial, resulting
from the combination of the biological characteristics of
the fungus and the immune status of the patient, wherein
the latter seems to be more important (Abad et al., 2010;
Balloy & Chignard, 2009; Tekaia & Latgé, 2005).
Aspergillosis is the general term that describes a wide
spectrum of diseases caused by Aspergillus spp. Such diseases
manifest themselves as non-invasive saprophytic or angioinvasive diseases (Hogan et al., 1996; Hope et al., 2005).
Angioinvasive diseases occur after conidia inhalation and
Abbreviations: CA, carbonic anhydrase; CAI, carbonic anhydrase
inhibitor; HCR, high CO2-requiring; IA, invasive aspergillosis.
064444 G 2014 SGM
alveolar infection. Once hyphae are formed, invasion of the
pulmonary vascular endothelial cells develops (a process
called angioinvasion), and an extensive dissemination to
other organs through the bloodstream may be established
(Filler & Sheppard, 2006; Hope et al., 2005; Pfaller &
Diekema, 2010).
Several species of Aspergillus such as A. flavus, A. niger, A.
terreus and A. versicolor can cause aspergillosis, but A.
fumigatus reigns as the main causative agent. It was
responsible for 90 % of the invasive aspergillosis (IA) cases
in the 1980s, for 50–60 % between the 1990s and 2000s,
and for 72.6 % between 2004 and 2008 (Pfaller & Diekema,
2010; Steinbach et al., 2012).
A. fumigatus infection can cause allergic bronchopulmonary aspergillosis in immunologically hypersensitive individuals, chronic pulmonary aspergillosis in patients with
pulmonary disorders and IA in immunocompromised
patients, which represents the most severe and lifethreatening disease form, with a 40–90 % mortality rate
(Fortún et al., 2012; Lin et al., 2001). The most frequent
site of IA infection is the lung (Steinbach et al., 2012), but
IA also has clinical forms of extrapulmonary dissemination
manifested throughout the host bones (Winterstein et al.,
2010), joints (Yu et al., 2010), heart (Kalokhe et al., 2010),
brain (Thakar et al., 2012), oesophagus (Akyol Erikci et al.,
2009), stomach, liver (Scott et al., 2007), intestine (Mohite
et al., 2007), eyes (Pushker et al., 2011), kidneys (Jung et al.,
2007) and skin (cutaneous aspergillosis) (Tahir et al., 2011)
(Fig. 1).
It is important to mention that invasive cutaneous
aspergillosis is characteristic of primary and secondary
infections. In primary cutaneous aspergillosis, the infection
starts through contact of contaminated material in
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15
J. M. Tobal and M. E. S. F. Balieiro
Extrapulmonary
dissemination
Bronchopulmonary
aspergillosis
An
gio
inv
as
ion
Inhalation
Airborne
conidia
n
Traumatized skin
sio
o
gi
An
a
inv
B
l
o
o
d
s
t
r
e
a
m
Primary cutaneous
aspergillosis
Secondary cutaneous
aspergillosis
Bones, joints, heart, brain,
oesophagus, stomach, liver,
intestine, eyes, kidney, etc.
Fig. 1. Clinical forms of aspergillosis. Parallel between primary and secondary cutaneous aspergillosis, bronchopulmonary
aspergillosis and the process of angioinvasion leading to extrapulmonary dissemination.
traumatized skin (i.e. intravenous and central access,
occlusive dressings, burns and surgical wounds). In a
neutropenic host, angioinvasion can occur after the
epidermis is disrupted, which can lead to vessel thrombosis, tissue necrosis and dissemination (Walsh, 1998). In
secondary cutaneous aspergillosis, the infection occurs
through haematogenous dissemination from another
contaminated tissue source (Fig. 1). Although considered
rare, some recent outbreaks of cutaneous aspergillosis have
been reported (Kim et al., 2010a; Tahir et al., 2011; van
Burik et al., 1998).
Immunocompromised populations that are more susceptible to IA include patients with haematological malignancy,
solid organ and haematopoietic stem cells, transplant
recipients, patients with a solid tumour, human immunodeficiency virus/AIDS patients, patients with inherited
immunodeficiency disorders and others (Steinbach et al.,
2012). Among these, special attention is given to transplant
recipients since they are the ones at highest risk of fungal
infections (Neofytos et al., 2009, 2010; Steinbach et al.,
2012). In addition, cytomegalovirus disease, graft-versushost disease, advanced age, poor transplant function, use of
corticosteroids, episodes of protein malnutrition, uraemia,
hyperglycaemia, leukopenia, use of tubes or catheters, and
multiple or acute rejection episodes are the factors that can
16
increase the risk of fungal infection in these patients (AsanoMori, 2010; Patel & Paya, 1997).
Other than the patient condition, another important risk
factor is the presence of Aspergillus spp. in the hospital
environment. Nosocomial aspergillosis can occur as a
result of increasing airborne conidia and water contamination, especially during hospital renovation and construction periods (Haiduven, 2009; Vonberg & Gastmeier,
2006). Although prophylactic efforts such as the use of
high-efficiency particulate air filtration have shown
encouraging results in decreasing nosocomial aspergillosis,
infection in the hospital setting still remains a risk for
transplant patients and further measures need to be taken
(Eckmanns et al., 2006; Garnaud et al., 2012).
IA treatment
Fungi have emerged as a major cause of human disease,
especially among patients who are immunocompromised
or hospitalized with serious underlying diseases. Although
the number of invasive fungal infections has increased
substantially recently, the number of antifungal drugs with
novel targets, developed over the past few decades, has been
restricted to the echinocandins class (Pfaller & Diekema,
2010; Shapiro et al., 2011).
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Journal of Medical Microbiology 63
Aspergillus fumigatus and carbonic anhydrases
Since A. fumigatus is the main pathogen involved with IA,
we will provide a brief discussion about the therapy for this
disease and its drug resistance data.
The sites of action of the available antifungal drugs include
cell-wall synthesis, membrane function, ergosterol synthesis, nuclear division and nucleic acid synthesis (Shapiro
et al., 2011). Among these, the drugs approved by the US
Food and Drug Administration for treatment of IA are
amphotericin-deoxychlate, amphotericin lipid formulations [amphotericin B lipid complex (ABLC), liposomal
amphotericin B (L-AMB) and amphotericin B colloidal
dispersion (ABCD)], itraconazole, voriconazole, posaconazole and the most recent, the echinocardin caspofungin
(VandenBussche & Van Loo, 2010; Walsh et al., 2008).
Conventional polyenes (amphipathic drugs that bind
strongly to ergosterol, disrupting the fungal cell membrane,
e.g. amphotericin-deoxycholate) and the first generation of
triazoles (antifungals that inhibit the synthesis of ergosterol
by inhibiting the enzyme lanosterol 14 a-demethylase, e.g.
itraconazole) are effective against several fungal pathogens,
and these drugs were widely used some years ago.
However, the toxicity of polyenes and the emergence of
resistance among the triazoles have been limiting the use of
these drugs (Bowyer et al., 2011; da Silva Ferreira et al.,
2004; Denning, 1998; Jeurissen et al., 2012; Klepser, 2011).
Recent IA therapeutic studies have shown good results with
the use of the second-generation triazoles voriconazole and
posaconazole. Voriconazole has an excellent activity
against Aspergillus spp. and is approved for IA first-line
treatment, while posaconazole is approved for prophylaxis
of invasive fungal infections and second-line treatment of
IA (Karthaus, 2011; Messer et al., 2006; Walsh et al., 2008).
Unfortunately, the appearance of A. fumigatus isolates that
are less susceptible to several azoles, including the highspectrum examples such as voriconazole and posaconazole,
is increasing (Arendrup et al., 2008; Howard et al., 2006;
Shapiro et al., 2011). This can be explained by the fact that
they are the only agents available in oral form, being
frequently used in prophylaxis and chronic infection
(Howard & Arendrup, 2011; Manavathu et al., 2001).
Also, azole exposure in the environment through azole
fungicides, for example, can contribute to multi-azole
resistance (Snelders et al., 2012; Verweij et al., 2009).
Use of lipid formulations of amphotericin B (ABLC and LAMB) are also considered as an alternative primary therapy
for some patients (Cornely et al., 2007; Herbrecht et al.,
2002; Patterson et al., 2005; Walsh et al., 2008). These
drugs have lower nephrotoxicity and infusion-related
reactions compared to conventional amphotericin B
(amphotericin-deoxychlate), which was the considered
‘standard therapy’ for IA (Barcia, 1998; Deray, 2002;
Ostrosky-Zeichner et al., 2003; White et al., 1998).
Besides the triazoles and polyenes, a third treatment option
for IA is the use of echinocandins (e.g. caspofungin). This
class of drugs has its target as fungal cell-wall synthesis by
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inhibiting b-(1,3)-D-glucan synthase, and it seems to be a
well-tolerated drug with rare resistant Aspergillus specimens (Kartsonis et al., 2003). However, it is not considered
for monotherapy since it only has fungistatic activity
against Aspergillus spp. Instead, it is considered an option
when patients present with voriconazole-refractory aspergillosis or if they are intolerant to the other therapies
(Denning et al., 2006; Maertens et al., 2004).
As opposed to azoles, little is known about the resistance of
echinocandins. The MIC test is not considered an
appropriate sensible test to evaluate the susceptibility of
Aspergillus spp. to echinocandins. For susceptibility
evaluation, the minimum effective concentration test seems
to show better results for Aspergillus spp. However, it is still
unclear how these results can be correlated with the
treatment success of echinocandins, and resistance may be
underdiagnosed due to technical limitations (Arendrup
et al., 2008; Mayr et al., 2012). In addition, unusual IA
outbreaks in patients receiving caspofungin therapy and
the selection pressure caused by its use, presume possible
future echinocandin resistance (Madureira et al., 2007;
Mayr & Lass-Flörl, 2011; Phai Pang et al., 2012).
Carbonic anhydrases (CAs)
CAs (EC 4.2.1.1) were first studied in erythrocytes in 1933
(Meldrum & Roughton, 1933; Stadie & O’Brien, 1933),
being the first enzymes known to contain zinc in their
active site. These enzymes are widely found in all life
kingdoms (Eukarya, Bacteria and Archaea) playing important roles in the global carbon cycle (Ferry, 2013). CAs
catalyse a simple but essential physiological reaction: the
hydration of CO2 to bicarbonate (HCO32) and the
corresponding dehydration of HCO32 in acidic medium
with regeneration of CO2. In high concentrations of CO2,
this reaction can occur spontaneously; however, thanks to
these enzymes, this reaction is much facilitated (Supuran
et al., 2003; Supuran, 2008a, 2010).
The rate of enhancement is about 10 000 times the natural
rate of CO2 hydration, which is very important because of
the physiological roles of CO2 and HCO32 (Lindskog,
1997). CO2 and HCO32 are substrates and products in
several reactions, participating in processes that must be
rapid, such as transport and metabolic processes (Berg
et al., 2002; Imtaiyaz Hassan et al., 2013; Supuran et al.,
2003).
CO2 is used by autotrophic organisms for energy
production, whereas it is a product of aerobic metabolism
in heterotrophic organisms. In heterotrophic organisms,
CO2 is inhaled, released into the blood and transported to
the lungs for exhalation. In the blood, CO2 reacts with
water forming carbonic acid, which further becomes a
bicarbonate ion and a proton. In this manner, the CAs are
involved in several physiological and pathological processes
throughout the different kingdoms of life including CO2
and pH homeostasis, CO2/HCO32 transport in various
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J. M. Tobal and M. E. S. F. Balieiro
metabolizing tissues, respiration, bone calcification, retina
and nervous system electric activity, CO2 fixation, biosynthetic reactions (gluconeogenesis, lipogenesis and ureagenesis), electrolyte secretion and cancer development (Bahn &
Mühlschlegel, 2006; Henry, 1996; Imtaiyaz Hassan et al.,
2013; Potter & Harris, 2003; Schlicker et al., 2009; Supuran
& Scozzafava, 2007; Supuran, 2008b; Tripp et al., 2001).
The CAs evolved independently in five genetically distinct
enzyme classes. All classes are metalloenzymes that use
metal ions at the active site (Supuran et al., 2003). The aCAs, which use Zn2+ ions at the active site, are normally
monomers and rarely dimers. They are encountered in
vertebrates, the cytoplasm of green plants, protozoa, algae,
fungi and in some bacteria; there are 16 isoforms of a-CAs
already found in mammals (except primates that possess
only 15 CAs) (Cuesta-Seijo et al., 2011; Elleuche &
Pöggeler, 2009a; Supuran, 2008a, 2010). The b-CAs, which
use Zn2+ ions at the active site, are dimers, tetramers or
octamers, and they are present in bacteria, fungi, algae,
plants and some Archaea. The c-CAs probably use Fe2+
ions at the active site, but they are also active with bound
Zn2+ or Co2+ ions. The c-CAs are trimers and are found
in Archaea and bacteria; it is postulated that the c-CA class
evolved as an ancient enzyme playing an important role in
the metabolism of early life (Rowlett, 2010; Supuran,
2008a, 2010). The d-CAs, which use Zn2+ ions at the active
site, are probably monomers and are present in marine
diatoms. Similarly, the f-CAs, which use Cd2+ or Zn2+
ions at the active site, are probably monomers, and they are
found in marine diatoms and cyanobacteria (Bahn &
Mühlschlegel, 2006; Cox et al., 2000; Ferry, 2013; Ferry &
House, 2006; Krungkrai & Supuran, 2008; Lane & Morel,
2000; Liljas & Laurberg, 2000; So et al., 2004; Tripp et al.,
2001; Xu et al., 2008).
As discussed above, fungal CAs belong mostly to the bclass, but some a-CAs have been found in these microorganisms as well. A genomic search study identified a-CAs
in filamentous ascomycetes, with the exception of hemiascomycetous yeasts and basidiomycetes. Surprisingly, this
search was able to find a-CA genes in species of Aspergillus
such as A. terreus, A. oryzae, A. flavus and A. niger.
However, the same was not observed for A. fumigatus, A.
nidulans and A. clavatus (Elleuche & Pöggeler, 2009a)
(Table 1, Fig. 2).
In fungi, the CAs seem to be located mainly in the
cytoplasm and mitochondria. In silico analysis predicts the
localization of two CAs in the mitochondria and another
two in the cytoplasm of A. fumigatus. In A. nidulans, the
single pair of CAs is predicted to be located only in the
cytoplasm (Han et al., 2010). The existence of CAs with or
without an N-terminal mitochondrial target is related to
the duplication of genes during the evolution of filamentous ascomycetes fungi. It seems that the mitochondrial
localization occurred after duplication of the genes and the
appearance of isoforms, suggesting also that the translocation of the isoforms into the mitochondria may denote the
18
gain of novel mitochondrial-specific functions (Elleuche &
Pöggeler, 2009a). The mitochondrial location of fungal bCAs was shown for the first time in the ascomycetous
fungus, Sordaria macrospora. Three b-CA isoforms were
found in this fungus; the cytoplasmic isoforms CAS1 and
CAS3, and the mitochondrial CAS2, which was proven to
be required for ascospore germination and vegetative
growth (Elleuche & Pöggeler, 2009b).
CO2 sensing and metabolism via CAs play important roles
in the proliferation, survival and differentiation of diverse
pathogenic fungi infecting human hosts. The reason for
this is the drastic difference in CO2 levels in an infected
host (5 % CO2) and in the natural habitat (0.033–0.038 %
CO2) (Bahn et al., 2005; Bahn & Mühlschlegel, 2006;
Innocenti et al., 2008, 2010; Isik et al., 2008; Klengel et al.,
2005; Krungkrai & Supuran, 2008; Mogensen et al., 2006;
Rowlett, 2010; Supuran & Scozzafava, 2007).
The NCE103 gene encodes a plant-like b-CA in Saccharomyces cerevisiae, and it was shown to be present in several
fungal species, including the pathogenic Candida albicans,
Candida glabrata and Cryptococcus neoformans (Bahn et al.,
2005; Elleuche & Pöggeler, 2010; Innocenti et al., 2009a, b;
Klengel et al., 2005). Its inactivation in Saccharomyces cerevisiae (deletion mutant Dnce103) results in a
high CO2-requiring (HCR) mutant, suggesting that a
functional CA is an important prerequisite for
Saccharomyces cerevisiae to grow under low CO2 concentrations. Furthermore, in lower concentrations of CO2,
there is an increase in the CA expression in Saccharomyces
cerevisiae (Amoroso et al., 2005; Götz et al., 1999).
Likewise in Saccharomyces cerevisae, b-CA deletion mutants
of Candida albicans and Cryptococcus neoformans have the
HCR phenotype, not being able to grow in the atmospheric
concentration of CO2. Both Candida albicans and Cryptococcus neoformans seem to be similar in that their adenylyl
cyclase is sensitive to physiological concentrations of CO2/
HCO32, suggesting that the link between cAMP signalling
and CO2/HCO32 sensing is a conserved mechanism between
the fungal species (Klengel et al., 2005; Mogensen et al.,
2006).
Cryptococcus neoformans, a fungal pathogen of humans
that causes fatal meningitis in immunosuppressed
patients, has two potential CA homologues (Can1 and
Can2), although only Can2 plays essential roles during
both cellular growth in environmental ambient conditions
and sexual differentiation of the pathogen (Bahn et al.,
2005). High CO2 concentrations (5 %), encountered in
infected hosts, induce capsule biosynthesis, an important
virulence factor for this pathogen, through activation of
adenylyl cyclase by bicarbonate (Mogensen et al., 2006).
Furthermore, transcriptional analysis of Cryptococcus
neoformans has demonstrated that CA-dependent genes
are involved in fatty acid biosynthesis, organization of the
polysaccharide capsule, sexual differentiation, the environmental stress response and the oxidative stress
response (Kim et al., 2010b).
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Aspergillus fumigatus and carbonic anhydrases
Table 1. a- and b-CAs of Aspergillus spp., Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans and Sordaria
macrospora
Species
Aspergillus clavatus NRRL 1
Aspergillus flavus NRRL3357
Aspergillus fumigatus Af293
Aspergillus nidulans FGSC A4
Aspergillus niger CBS 513.88
Aspergillus oryzae RIB40
Aspergillus terreus NIH2624
Neosartorya fischeri NRRL 181§
Saccharomyces cerevisiae S288c
Candida albicans WO-1
Cryptococcus neoformans var. grubii H99
Sordaria macrospora
NCBI Protein accession no.*D
Region named
EAW12033.1
EAW12034.1
EAW10562.1
XP_002374234.1
XP_002378947.1
XP_002384772.1
XP_751704.1
XP_001481412.1
XP_751882.1
XP_001481413.1
CBF81553.1
CBF85595.1
XP_001398618.1
XP_001390254.2
XP_001396389.1
BAE58191.1
BAE66418.1
XP_003190338.1
EAU33866.1
EAU36091.1
XP_001209372.1
EAU38812.1
XP_001213134.1
XP_001262181.1
XP_001267068.1
XP_001266926.1
NP_014362.3
EEQ43380.1
EEQ44200.1
AFR93887.2
AFR94409.1
CAX51127.1
CAT00780.1
CAT00781.1
CAT00782.1
b-CA clade A
b-CA
b-CA clade A
b-CA clade A
b-CA clade D
a-CA prokaryotic-like
b-CA clade A
b-CA clade A
b-CA clade D
b-CA
b-CA clade A
b-CA clade A
b-CA
b-CA clade A
a-CA prokaryotic-like
b-CA clade A
a-CA prokaryotic-like
b-CA clade D
a-CA prokaryotic-like
a-CA
b-CA clade D
a-CA prokaryotic-like
b-CA clade A
b-CA clade A
b-CA clade D
b-CA clade A
b-CA clade A
b-CA clade D
b-CA clade A
b-CA clade A
b-CA clade A
a-CA
b-CA clade A
b-CA clade A
b-CA clade D
*NCBI Protein accession numbers or numbers of hypothetical proteins from genome projects.
DOnly b-CAs were used for generation of the phylogenetic tree.
dClade A: plant-like b-CAs; clade D: cab-like b-CAs.
§Teleomorph of A. fischerianus (a very close homothalic sexual relative to A. fumigatus).
Physiological levels of CO2 induce filamentous growth, an
important virulence factor, and promote white to opaque
switching in Candida albicans, thus facilitating mating by
activation of the transcription factor Flo8 (Du et al.,
2012). The activation of transcription factor Flo8 can
occur by direct stimulation of adenylyl cyclase activity or
by another pathway, independent of adenylyl cyclase. In
addition, CA is essential for the pathogenesis of Candida
albicans in niches where the available CO2 is limited or
not supplied by the host. The skin is an example of a
biologically limited CO2 niche. On the skin, the
concentration of CO2 is lower than inside the host
because of the equilibrium with the atmospheric CO2
concentration (Allen & King, 1978). During Candida
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albicans skin infection, CA seems to play a role in the
epithelial invasion by this fungus (Klengel et al., 2005).
The variation of CO2 concentration in niches where
Candida albicans is able to grow and infect reveals the
importance of the transcription factor Rca1p, the first
direct CO2 regulator of CA in yeast. The transcription
factor Rca1p activates CA at lower concentrations of CO2
in Candida albicans, independent of the adenylyl cyclase
and also seems to repress virulence-related genes, confirming the existence of a cAMP-independent CO2
signalling pathway. Thus, the CO2 sensing in fungi
appears to be accomplished by combined pathway routes,
and the relationship between them remains to be unveiled
(Cottier et al., 2012).
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J. M. Tobal and M. E. S. F. Balieiro
98 A. fumigatus XP_001481412.1
93
0.2
82
N. fischeri XP_001262181.1
A. clavatus EAW12033.1
98
A. nidulans CBF85595.1
38
S. macrospora CAT00781.1
40
S. macrospora CAT00780.1
C. neoformans AFR93887.2
39
94
15
C. neoformans AFR94409.1
A. niger XP_001398618.1
A. clavatus EAW10562.1
11
98
15
A. fumigatus XP_751704.1
95 N. fischeri XP_001266926.1
A. nidulans CBF81553.1
15
A. niger XP_001390254.2
85
A. terreus XP_001213134.1
A. flavus XP_002374234.1
92 A. oryzae BAE58191.1
A. clavatus EAW12034.1
97
A. fumigatus XP_001481413.1
81
S. cerevisiae NP 014362.3
96
C. albicans EEQ44200.1
99 A. fumigatus XP_751882.1
37
65
N. fischeri XP_001267068.1
C. albicans EEQ43380.1
99
S. macrospora CAT00782.1
A. oryzae XP_003190338.1
49
85
A. terreus XP_001209372.1
A. flavus XP_002378947.1
Fig. 2. Phylogenetic tree of the zinc-coordinating region from fungal b-CAs. This tree was constructed by the neighbour-joining
method. The topology was also evaluated by bootstrap analysis (MEGA5.2; 1000 repeats). The numerical values in the trees
represent bootstrap results. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary
distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method;
the bar indicates the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were
eliminated only in pairwise sequence comparisons (pairwise deletion option). Fungal protein sequences used for this figure are
available at the National Center for Biotechnology Information (NCBI: http://www.ncbi.nlm.nih.gov/protein/). All downloads
were performed before 25 September 2013.
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Aspergillus fumigatus and carbonic anhydrases
The level of Rca1p transcripts in hypercapnia is twice as
high as the level at atmospheric CO2 concentrations, while
Rca1p orthologues in Saccharomyces cerevisiae and Candida
glabrata, Cst6p and CgRca 1p, respectively, present no
variation in expression between the different levels of CO2
concentration (Cottier et al., 2012, 2013). This expected
evolutionary pliancy is also observed in other regulatory
complexes between species (Lavoie et al., 2010). In all
species, however, inactivation of the transcription factor
results in CA induction loss in ambient CO2 levels.
A. fumigatus is also a pathogen that faces dramatic changes
in CO2 concentration during the infection process. Four
isoforms of b-CAs have been identified in A. fumigatus,
namely cafA–D. Both cafA and cafB are constitutively and
strongly expressed, while cafC and cafD are weakly expressed
and are CO2 inducible. Similar to other pathogens discussed
above, the double mutant DcafADcafB is not able to grow in
ambient CO2 conditions, presenting an HCR phenotype.
This phenotype is reversed when in an environment of 5 %
CO2 (Han et al., 2010). Furthermore, defects of CAs affect A.
fumigatus conidiation, which is in accord with results shown
in Saccharomyces cerevisiae, where the CA seems to be
involved in spore formation (Jungbluth et al., 2012). When
single and double mutants of CAs were tested in a low-dose
murine infection, the virulence was not affected, which was
also observed with other pathogens. However, a possible role
of CAs on A. fumigatus virulence has not been discarded
since triple and quadruple deletion mutants of the CAs were
not constructed (Han et al., 2010).
CA inhibitors (CAIs)
Sulfonamides were the first CAIs described. Since then,
other chemical substances with similar activity have also
started to be evaluated for prevention or therapeutic use as
anti-convulsants, anti-glaucoma agents, vasodilators and
diuretics (Friedberg et al., 1953; Gloster & Perkins, 1955;
Gray et al., 1957; Millichap et al., 1955). Current studies
suggest the potential use of CAIs as anti-cancer (Husain &
Madhesia, 2012; Winum et al., 2012), anti-obesity and
anti-pain agents (Supuran, 2010, 2011).
The cloning of the genome of many pathogens allows the
search for anti-infective agents with a novel mechanism of
action (diverse mechanism of action compared with
clinically used drugs for which drug resistance was reported)
through exploration of alternative routes for inhibiting
proteins essential for the life cycle of the pathogens or for
virulence (Del Prete et al., 2012; Supuran, 2011).
The identification of CAs in known micro-organisms such
as Bacillus anthracis (Wilson et al., 2008), Helicobacter
pylori (Morishita et al., 2008), Plasmodium falciparum
(Krungkrai & Supuran, 2008), Mycobacterium tuberculosis
(Nishimori et al., 2009), Haemophilus influenzae (Cronk
et al., 2006), Vibrio cholerae (Del Prete et al., 2012),
Salmonella enterica (Nishimori et al., 2011), Brucella suis
(Joseph et al., 2010, 2011), Streptococcus pneumoniae
http://jmm.sgmjournals.org
(Burghout et al., 2010), Candida albicans (Innocenti et al.,
2008; Klengel et al., 2005), Candida glabrata (Innocenti
et al., 2010), Cryptococcus neoformans (Bahn et al., 2005;
Innocenti et al., 2008; Klengel et al., 2005; Mogensen et al.,
2006), Saccharomyces cerevisiae (Isik et al., 2008), Sordaria
macrospora (Elleuche & Pöggeler, 2009a), A. fumigatus and
A. nidulans (Han et al., 2010) has been driving studies of
effectiveness and performance of CAIs as anti-infective
agents.
P. falciparum presents decreased growth in the presence of
CAIs. This important parasite, which is one of the
responsible agents of malaria, has a purine and pyrimidine
requirement during its intra-erythrocytic development.
The pathway for pyrimidine synthesis in the host differs
from the pathway in the parasite, being a desirable target
for drug development. It is the CAs that catalyse the
formation of bicarbonate, which is a substrate for the
parasitic pyrimidine pathway. A ureido-sulfonamide derivative is an example of a CAI that was shown to be effective
against in vitro pathogen growth (Krungkrai et al., 2008;
Krungkrai & Supuran, 2008; Supuran, 2010).
In the case of H. pylori, a pathogen associated with
gastroduodenal diseases, there are two classes of CA
characterized: hpaCA (a-CA) and hpbCA (b-CA). CAs
are necessary for H. pylori survival in the stomach, being
associated with acid acclimation, urea and bicarbonate
metabolism of this pathogen (Marcus et al., 2005; Stähler
et al., 2005). Supiride, a benzamide derivative, presents in
vitro and in vivo inhibitory activity against H. pylori a- and bCAs and was effective in killing first- and second-line therapyresistant strains (Morishita et al., 2008). Sulfonamides/
sulfamates are also able to strongly inhibit the b-CA of H.
pylori, and a group of anions and molecules that interact with
zinc proteins have also shown inhibitory activity against
hpaCA and hpbCA, indicating that CAIs can be a possible
alternative for gastric disease therapy (Maresca et al., 2013;
Nishimori et al., 2007).
The CAs of M. tuberculosis, a pathogen with a high number
of resistant strains, have also been shown to be effectively
inhibited by sulfonamides/sulfamates (Nishimori et al.,
2009, 2010), and a new C-cinnamoyl glycoside containing
the phenol moiety was the first CAI with anti-tubercular
activity (Buchieri et al., 2013).
In fungal organisms, different chemotypes of CAIs have
already been tested against b-CAs of Saccharomyces
cerevisiae, Cryptococcus neoformans, Candida albicans,
Candida glabrata and Malassezia globosa. Some of the
compounds also show inhibitory activity against a-CAs,
indicating the need for more selective b-CA inhibitors
(Hewitson et al., 2012; Innocenti et al., 2009a, 2009b;
Monti et al., 2012). In conformity with CA characterization
studies, the sulfonamide inhibitor, ethoxzolamide, significantly reduces the growth of Cryptococcus neoformans and
Candida albicans at ambient levels of CO2 (Klengel et al.,
2005; Mogensen et al., 2006). In addition, M. globosa and
other dermatophytic fungi have demonstrated fragmented
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21
J. M. Tobal and M. E. S. F. Balieiro
hyphae under sulfonamide treatment. Results are comparable to treatment with ketoconazole, a clinically used
antifungal agent (Hewitson et al., 2012).
To our knowledge, there is no CAI study for A. fumigatus.
Libraries of compounds for inhibition screening and highresolution X-ray crystal structures of possible related b-CAs
are available. These methods help to identify potential
specific inhibitors for drug development based on the
interaction and structural analysis at a molecular level.
Having said that, homology modelling studies could be
done for A. fumigatus in order to bring a better
understanding of its CAI profiles, like that performed for
Nce103 (Candida albicans) based on the crystal structure of
Can 2 (Cryptococcus neoformans) (Innocenti et al., 2009a;
Schlicker et al., 2009).
In Fig. 3, we have shown the alignment of Cryptococcus
neoformans and Candida albicans CA amino acids, reported
in the literature, with the four A. fumigatus CAs, cafA–D.
The zinc ligand Cys-His-Cys is conserved in all of them,
showing their relatedness to the b-class. In a- and c-CAs,
zinc is bound to three conserved His residues. Asp/Arg
pairs are also present in most of the b-CAs analysed. It is
known that the fourth zinc coordination site allots its
catalytic cycle in two different ways. In some b-CAs, either
a water molecule or acetate is found at the fourth zinc
coordination site; other b-CAs possess a conserved Asp in
that region (Tripp et al., 2001).
b-CAs can also be classified into two subclasses: ‘plant-
type’ or ‘cab-type’ (Table 1). It seems that cafA
(XP_751704.1) and caf B (XP_001481412.1) are plant-like
A. fumigatus_XP_751882.1
C. albicans_EEQ43380.1
C. albicans_EEQ44200.1
A. fumigatus_XP_001481413.1
A. fumigatus_XP_751704.1
A. fumigatus_XP_001481412.1
C. neoformans_AFR93887.2
C. neoformans_AFR94409.1
120
60
180
240
300
Fig. 3. Protein alignment for b-CAs from Candida albicans (NCBI Protein nos EEQ44200.1 and EEQ43380.1), Cryptococcus
neoformans Can1 (AFR93887.2) and Can2 (AFR94409.1), Aspergillus fumigatus cafA (XP_751704.1), cafB
(XP_001481412.1), cafC (XP_751882.1) and cafD (XP_001481413.1). Sequences were aligned using ClustalO (http://
www.ebi.ac.uk/Tools/msa/clustalo/) and the Pearson/FASTA output was analysed using Protein Boxshade (http://www.fr33.net/
boxshadeprotein.php) to show identities (.50 %, black background) and similarities (.50 %, grey shading). Zn+2-binding
amino acids are shown in red, Asp–Arg conserved pairs are shown in blue and plant-type amino acids are shown in yellow.
Thirty amino acids were added at the N terminus of cafD, according to cDNA analysis by Han et al. (2010).
22
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Journal of Medical Microbiology 63
Aspergillus fumigatus and carbonic anhydrases
b-CAs (clade A), cafC is a cab-like b-CA (clade D) and the
subclass of caf D is unknown (Elleuche & Pöggeler, 2009a).
Design of inhibitors has to consider the differences between
the catalytic sites of b-CAs, since the affinity for the
inhibitors seems to vary (Tripp et al., 2001).
In A. fumigatus, the lack of virulence changes in murine
models by the infection of each of the four deleted b-CA
mutants, and the appearance of the same HCR phenotype as
Saccharomyces cerevisiae, Candida albicans and Cryptococcus
neoformans in the double mutant DcafADcafB, suggests the
existence of conserved pathways of CO2 sensing on fungal
pathogens that face changes in CO2 concentration during
infection. The CA enzymes of these pathogens seem to play a
role when CO2 is not supplied at satisfactory levels by the
host. It is important to highlight again that the role of CAs in
the pathogenicity of A. fumigatus is not yet conclusive, since
triple and quadruple deleted mutants were not constructed
by more recent available studies (Han et al., 2010; Klengel
et al., 2005; Mogensen et al., 2006).
As previously mentioned, A. fumigatus has four CAs
belonging to two different subclasses, which could make
the search for therapeutic CAIs to this organism a very
challenging task. Yet, the previous steps still need to be
taken to understand more about the pathways that might
relate to CA/CO2 for this fungus, leading to the answers of
the following questions: Is there a conserved pathway
between the pathogens that face changes of CO2 during the
infection? Do A. fumigatus CAs also play a role when CO2
is not supplied in satisfactory levels by the host during the
infection process? If yes, would CAs be an important factor
during primary cutaneous aspergillosis infection as
observed in skin infections caused by Candida albicans?
How do the different CAs interact with other metabolic
pathways during the physiopathology of diseases caused by
A. fumigatus? Would CAIs be effective as a synergistic and/
or prophylactic drug in cases of aspergillosis in tissues
where the CO2 concentration is limited, such as the eyes
and the skin?
Despite these currently unanswered questions, the fact that
several pathogenic micro-organisms such as A. fumigatus
possess b-CA enzymes, which are not present in humans,
increases the expectation of future studies with CAIs
aiming to develop novel anti-infective agents.
yet completely understood. Given the existing antifungal
resistance data and the search for new drug targets, whether
the CAIs are drugs that could be considered as new antiinfective drugs remains to be evaluated. By the same token,
based on the studies presented in this review, we also
expect that CAs will still deserve to be suitable subject
matter in further studies involving human pathogens.
Acknowledgements
We would like to thank the Foundation for Research Support of the
State of São Paulo and the National Council for Scientific and
Technological Development, both agencies from Brazil, for supporting our research. The authors report no conflicts of interest.
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