text:
Experimental medical mycological research in Latin America - a 2000-2009 overview.
ABSTRACT An overview of current trends in Latin American Experimental Medical Mycological research since the beginning of the 21(st) century is done (search from January 2000 to December 2009). Using the PubMed and LILACS databases, the authors have chosen publications on medically important fungi which, according to our opinion, are the most relevant because of their novelty, interest, and international impact, based on research made entirely in the Latin American region or as part of collaborative efforts with laboratories elsewhere. In this way, the following areas are discussed: 1) molecular identification of fungal pathogens; 2) molecular and clinical epidemiology on fungal pathogens of prevalence in the region; 3) cell biology; 4) transcriptome, genome, molecular taxonomy and phylogeny; 5) immunology; 6) vaccines; 7) new and experimental antifungals.
Article: Susceptibility to caspofungin of Candida spp. strains isolated in Ceará, Northeastern Brazil
C. Rocha da Silva, R. de Sousa Campos, M. Adalgiza dos Santos Neta, M. Rozellê Ferreira Ângelo, H. Iury Ferreira Magalhães, B. Coêlho Cavalcanti, M. Odorico de Moraes, D. Silveira Macedo, H. Vitoriano Nobre Júnior[Show abstract] [Hide abstract]
ABSTRACT: La caspofungine est une échinocandine prescrite pour le traitement des infections fongiques invasives causées par Candida sp. et Aspergillus sp. Le but de cette étude est d'évaluer le degré de sensibilité à la caspofungine de Candida sp., isolé à partir d'hémocultures dans l'état du Ceará (Brésil) par la méthode de microdilution. Trente-trois souches de Candida sp. ont été sélectionnées pour le test (sept C. albicans, neuf C. tropicalis et 17 C. parapsilosis) ; ces souches sont le plus souvent isolées dans des infections fongiques en Ceará. Les résultats des tests de sensibilité par microdilution peuvent être lus à 24 ou 48 heures après les tests, sans compromettre les interprétations du test. C. parapsilosis montre les CMI les plus élevées comparativement à celles de C. albicans et C. tropicalis pour la caspofungine.Journal de Mycologie Médicale/Journal of Medical Mycology 12/2011; · 0.40 Impact Factor - SourceAvailable from: Kelly Grace MagalhaesAldo Henrique Tavares, Kelly Grace Magalhães, Raquel Das Neves Almeida, Rafael Correa, Pedro Henrique Burgel, Anamélia Lorenzetti Bocca[Show abstract] [Hide abstract]
ABSTRACT: Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1β that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1β in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL-1 receptor, we demonstrated that IL-1β signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus.PLoS Neglected Tropical Diseases 12/2013; 7(12):e2595. · 4.49 Impact Factor - SourceAvailable from: Célia Maria de Almeida SoaresPatrícia Fernanda Zambuzzi-Carvalho, Patrícia Kott Tomazett, Suzana Costa Santos, Pedro Henrique Ferri, Clayton Luiz Borges, Wellington Santos Martins, Célia Maria Soares, Maristela Pereira[Show abstract] [Hide abstract]
ABSTRACT: The compound oenothein B (OenB), which is isolated from the leaves of Eugenia uniflora, a Brazilian Cerrado plant, interferes with Paracoccidioides yeast cell morphology and inhibits 1,3-beta-D-glucan synthase (PbFKS1) transcript accumulation, which is involved in cell wall synthesis. In this work we examined the gene expression changes in Paracoccidioides yeast cells following OenB treatment in order to investigate the adaptive cellular responses to drug stress. We constructed differential gene expression libraries using Representational Difference Analysis (RDA) of Paracoccidioides yeast cells treated with OenB for 90 and 180 min. Treatment for 90 min resulted in the identification of 463 up-regulated expressed sequences tags (ESTs) and 104 down-regulated ESTs. For the 180 min treatment 301 up-regulated ESTs and 143 down-regulated were identified. Genes involved in the cell wall biosynthesis, such as GLN1, KRE6 and FKS1, were found to be regulated by OenB. Infection experiments in macrophages corroborated the in vitro results. Fluorescence microscopy showed increased levels of chitin in cells treated with OenB. The carbohydrate polymer content of the cell wall of the fungus was also evaluated, and the results corroborated with the transcriptional data. Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment. The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile. Some of the changes may represent specific adaptive responses to this compound in this important pathogenic fungus.BMC Microbiology 10/2013; 13(1):227. · 2.98 Impact Factor
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Rev Iberoam Micol. 2011;28(1):1–25
Revista Iberoamericana
de Micología
Review article
Experimental medical mycological research in
Latin America - a 2000-2009 overview
Gioconda San-Blasa,∗, Eva Burgerb
aInstituto Venezolano de Investigaciones Científicas, Caracas, Venezuela
bUniversidade de São Paulo, Brazil
a r t i c l ei n f o
Article history:
Received 8 July 2010
Accepted 24 November 2010
Available online 15 December 2010
Latin America
Fungal pathogens
Cell biology
a b s t r a c t
An overview of current trends in Latin American Experimental Medical Mycological research since the
LILACS databases, the authors have chosen publications on medically important fungi which, according
research made entirely in the Latin American region or as part of collaborative efforts with laboratories
2) molecular and clinical epidemiology on fungal pathogens of prevalence in the region; 3) cell biology;
4) transcriptome, genome, molecular taxonomy and phylogeny; 5) immunology; 6) vaccines; 7) new and
experimental antifungals.
© 2010 Revista Iberoamericana de Micología. Published by Elsevier España, S.L. All rights reserved.
Investigación en micología médica experimental en América Latina-perspectiva
general entre 2000–2009
Palabras clave:
América Latina
Hongos patógenos
Biología Celular
Drogas Antifúngicas
r e s u m e n
en América Latina desde el inicio del siglo XXI (búsqueda bibliográfica desde enero de 2000 a diciembre
de 2009). Usando las bases de datos PubMed y LILACS, los autores hemos escogido publicaciones en
novedad, interés e impacto internacional, basadas en investigaciones realizadas totalmente en la región
latinoamericana o como parte de esfuerzos colaborativos con laboratorios de otras partes del mundo.
De esta forma, discutimos las siguientes áreas: 1) identificación molecular de patógenos fúngicos; 2)
epidemiología clínica y molecular de hongos patógenos prevalentes en la región; 3) biología celular; 4)
transcriptoma, genoma, taxonomía y filogenia moleculares; 5) inmunología; 6) vacunas; 7) antifúngicos
nuevos o experimentales.
© 2010 Revista Iberoamericana de Micología. Publicado por Elsevier España, S.L. Todos los derechos
Recent data206indicate that fungal diseases in Brazil do carry a
high toll on fatal outcome of systemic mycoses. Although Prado et
al's figures are limited to that country, it is reasonable to assume
that given the similarities in regional and local health services
and other social factors, their results may be representative of
∗Corresponding author.
E-mail address: email@example.com (G. San-Blas).
events in other Latin American countries. So, according to the
authors, death tolls in Brazil within the period 2005-2006, amount
to 44.6% (paracoccidioidomycosis, PCM), 26.8% (cryptococcosis),
(zygomycosis), and 0.8% (coccidioidomycosis) of total patients
treated for these diseases.206It is obvious, then, that Latin Ameri-
can countries suffer from a significant burden of systemic mycoses,
which need to be addressed not only in terms of public health poli-
1130-1406/$ – see front matter © 2010 Revista Iberoamericana de Micología. Published by Elsevier España, S.L. All rights reserved.
Page 2
G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
academic, experimental and clinical research aimed to understand
the phenomena underlying this serious and ever growing health
This review highlights publications chosen by the authors as
representative of the most relevant research in experimental med-
ical mycology carried out in Latin America in the period January
2000-December 2009. Seven areas are covered and discussed
within the framework of international research: 1) molecular
identification of fungal pathogens; 2) molecular and clinical epi-
demiology on fungal pathogens of prevalence in the region; 3)
cell biology; 4) transcriptome, genome, molecular taxonomy; 5)
immunology; 6) vaccines; 7) new and experimental antifungals.
Searching in PubMed and the Latin American LILACS data bases
(key words mycosis+<country> Jan 2000-December 2009), we
found 1958 publications assigned to Latin American countries,
either made entirely in the region or in collaboration with labora-
tories elsewhere, of which 1113 (57%) correspond to Brazil, 280 to
Mexico, 229 to Argentina, 88 to Colombia, 86 to Venezuela, and 44
to Chile. Paracoccidioides brasiliensis and PCM comprise 565 (29%)
of all regional publications in medical mycology, pointing to the
impact of this mycosis and its causal agent in Latin America, only
geographical region to host the fungus.229
Molecular identification of fungal pathogens
A major obstacle to the successful treatment of invasive fungal
infections is the paucity of rapid, sensitive and specific methods
that would help in the early diagnosis of fungal infections. PCR
assays for diagnostic purposes are being extensively used though
the method still lacks standardization and cannot be used as the
sole test for early detection or for the purpose of defining invasive
fungal infection [for a recent review, see 227].
In Latin America, PCR methodology for identification and diag-
nostic purposes has been applied ever since it appeared in the
scientific literature as a promising technique. The Histoplasma
capsulatum H or M antigens, pluripotent glycoproteins that elicit
both humoral and T cell-mediated immune responses, are pro-
teins whose genes have been used for the design of primers
aimed at molecular diagnosis.32,111Bracca et al32developed a
highly specific and sensitive semi-nested PCR assay in which three
antigen, were chosen for their ability to differentiate H. capsulatum
sequences from sequences of other fungal ß-glucosidases in the
databases. Meanwhile, Guedes et al111used the M-antigen gene
(highly homologous to catalases) to design four oligonucleotide
sequences in the less homologous regions, for application in a
sulatum. De Aguirre et al72used PCR technology in an enzyme
immunoassay format for the rapid differentiation of Aspergillus
species from other medically important opportunistic molds and
yeasts. With oligonucleotide probes, directed to the ITS2 region
of ribosomal DNA from several Aspergilli, they were able to dif-
ferentiate 41 isolates; a single DNA probe to detect all seven
of the most medically important Aspergillus species (Aspergillus
flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger,
Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor) was
also designed.
In a prospective study in a high-risk population of candidemia,
Moreira-Oliveira et al169used ITS5 and ITS4 primers for PCR, fol-
lowed by sequencing of products for Candida spp. identification.
They reported 72.1 and 91.2% sensitivity and specificity, respec-
tively. A combination of PCR and RFLP helped in the identification
of Candida species in immunocompromised and seriously dis-
observed, except for the group of newborns who were infected
the OPE-18 primer was reported to be a very specific and sensitive
method for the identification of Candida glabrata, Candida guillier-
mondii, Candida tropicalis, Candida pelliculosa, C. albicans, Candida
krusei, and Candida lusitaniae.14RAPD was also used for the correct
identification of C. albicans and Candida dubliniensis.20In 2006,
the latter species was detected for the first time in Venezuela by
means of a PCR approach.34A different methodological approach
monoclonal antibody, they were able to detect a 65-kDa antigen
against sera from patients: i) total clearance of antigenemia, ii)
initial clearance and relapse of antigenemia, and iii) partial clear-
ance of antigenemia. These results suggest that detection of the
65-kDa protein may be a valuable tool for the differential diagnosis
of candidemia caused by either of these Candida species.
The antigen 2/proline-rich antigen has been used for the PCR
detection of Coccidioides posadasii.26,71This antigen is common to
C. posadasii and Coccidioides immitis.
Diagnostic primers in P. brasiliensis have been designed pri-
marily from the nucleotide sequence of its reference antigen,
gp43.25,103Some of the gp43 sequences were taken from regions
where later studies164revealed the presence of informative and
non informative nucleotide substitutions in this highly polymor-
phic gene,164a result that may influence the use of gp43 as a
universal reference antigen.229A second set of diagnostic primers
tested in clinical samples is that of San-Blas et al.228They were
designed from two specific DNA fragments (Mw 0.72 and 0.83 Kb)
common to and specific for all P. brasiliensis samples, generated
when using the arbitrary primer OPG18 (Operon Biotechnology).37
from sputum and cerebrospinal fluid of PCM patients.228
A duplex polymerase chain reaction (PCR) targeting the ITS1-
5.8S-ITS2 region of the ribosomal DNA was designed for rapid and
specific identification of 69 Fonsecaea pedrosoi isolates; 4 Fonse-
not produced identification bands.73The frequency of Fonsecaea-
positive results was similar between duplex PCR (68.0%) and
morphology (67.0%). However, 4% isolates were positive by duplex
PCR but negative by morphology, indicating that PCR method may
be the test of choice when dealing with samples unable to produce
conidia. On the other hand, 3% samples were positive by morphol-
ogy and negative by Fonsecaea-specific PCR. These isolates have
high similarity to the genus Phialophora when DNA sequencing
analyses were performed.
Epidemiology of prevalent fungal pathogens in the region
Clinical epidemiology
Antimicrobial resistance surveillance serves for the detection
and tracking of resistance trends and emerging new resistance
threats, and also as a means to monitor the prevalent pathogens
causing serious infections. In order to address effectively any of
these objectives, the availability of a geographically diverse col-
lection of isolates from clinically important sites of infection is
essential.201Very few programs provide information on fungal
infections and antifungal resistance, among them, the ARTEMIS
Global Antifungal Susceptibility Program (ARTEMIS Program) and
the Regional Laboratory Network for Surveillance of Invasive Fun-
gal Infections and Antifungal Susceptibility in Latin America, both
mainly focused on candidemia from several Candida spp. Of recent
formation and consequently, few reports to date, the Regional
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
Table 1
Species distribution (%) of Candida bloodstream isolates in different Latin American regions
Geographical region/city/country
C. albicans C. parapsilosisC. tropicalis C. glabrata
Brazil, 9 cities
Caracas, Venezuela
BuenosAires, Argentina
Monterrey, Mexico
Network is coordinated by the Essential Medicines, Vaccines, and
with the technical and financial support of the National Center for
Microbiology of the Carlos III Health Institute (Spain), and the tech-
nical support of the Microbiology Department of the Dr. C. Malbrán
National Institute on Infectious Diseases (Argentina) and the
Microbiology Unit of the Parasitology Service of the Adolfo Lutz
Institute (Brazil).67The Network's main objectives are epidemio-
logical surveillance of invasive fungal infections through detection
of antifungal resistance and identification of emergent, invasive
fungal infections; establishment of norms and common proto-
cols for early diagnosis of mycoses; strengthening coordination,
countries.60The older ARTEMIS program was initiated in 2001
to provide focused surveillance of the activities of fluconazole
and voriconazole against Candida spp. causing invasive infections,
and to provide continuous development and validation of vari-
ous broth- and agar-based antifungal susceptibility test systems.
The ARTEMIS Program has provided a massive amount of data; it
uses a central reference laboratory and an international network of
105 participating centers as sources of clinical isolates201; several
Latin American laboratories provide essential information within
this program. Their results indicate that more than 90% of invasive
infections due to Candida spp. are attributed to five species, C. albi-
albicans remains the dominant species causing bloodstream infec-
from 37% in Latin America to 70% in Norway. Most notable is the
very low frequency of C. glabrata as a cause of BSI in Latin Amer-
ica, where only 4% to 7% of Candida BSIs are attributed to this
species.104,202The frequency of isolation of C. glabrata from blood
region (12.1% to 7.2%) between 2002 and 2004.169The frequency
of invasive candidiasis due to C. parapsilosis has increased in recent
years, most notably in Latin America. Whereas C. tropicalis is only
the fourth most common species of Candida causing BSI in North
America (7% of BSIs), it ranks second in Latin America (20%) and is
10-12%, respectively). C. guilliermondii and Candida rugosa are rela-
frequency as agents of invasive candidiasis. These two species can
be found in Latin America, where they each account for 3% to 5% of
all candidemias and may be more common than either C. glabrata
or C. krusei.60,78,104,109
The above mentioned global figures hide the documented geo-
graphic differences in rates and epidemiology of candidemia in
different countries and cities within them (Table 1). Prospective
candidemia surveillance in 11 medical centers located in 9 major
Brazilian cities (March 2003 to December 2004) indicated that C.
albicans was the most common species (40.9%), followed by C.
tropicalis (20.9%) and C. parapsilosis (20.5%).60Further candidaemia
surveillance studies in Brazil104indicated that in the genetically
heterogeneous taxon C. parapsilosis (C. parapsilosis, Candida orthop-
silosis and Candida metapsilosis), the prevalence of each species
among 141 bloodstream isolates was 88%, 9% and 3%, respectively.
All isolates but three 5-fluorocytosine-resistant C. orthopsilosis
were susceptible to polyenes, triazoles and caspofungin.
from January 2003 through August 2005, indicated that C. albicans
was the most frequently isolated yeast (46.7%), followed by C. trop-
icalis (19.0%), C. glabrata (9.2%) and C. parapsilosis (6.0%). During
the first year of an ongoing surveillance program of invasive fun-
gal infections in 13 hospitals in Chile, Silva et al234found that C.
albicans (40.8%), C. parapsilosis (13.1%), C. tropicalis (10%) and Cryp-
tococus neoformans (10%) were the most common yeast species. A
multicenter study performed to determine the species distribution
associated to candidemias in Argentina212also brought about sim-
ilar results, that is, C. albicans (40.75%), C. parapsilosis (28.67%), C.
tropicalis (15.84%), Candida famata (3.77%), C. neoformans (3.77%),
C. glabrata (2.64%), and others (4.53%). Interestingly, C. parapsilosis
a 3-year surveillance program (2004 to 2007) in Monterrey, Mex-
ico, followed by C. albicans (31.9%), C. tropicalis (14.8%), C. glabrata
(8.0%), among the most frequent. The species distribution differed
with the age of the patients, a result also documented by Pfaller
and Diekema.201
C. dubliniensis is associated with oral candidiasis in immunode-
pressed individuals. Using classical phenotypic methods combined
with PCR techniques, Jewtuchowicz et al114found that this species
is present in 4.4% of periodontal pockets of immunodepressed
Argentinean patients. C. albicans was the most frequent species,
corresponding to 24.4% (44/180). Other non-C. albicans species
were found, among them C. parapsilosis, C. tropicalis, and C. guil-
These studies were always accompanied by screening of resis-
tance to antifungals such as azoles and echinocandins, indicating a
wide range of variability in the susceptibility of strains. Together,
studies to guide physicians towards the most effective treatment
of candidiasis and other fungal diseases.
Retrospective studies characterizing acute/subacute PCM inci-
dence in the Botucatu area, São Paulo State, Brazil, from 1969
to 1999 and their relationship with climate variables (antecedent
precipitation, air temperature, soil water storage, absolute and rel-
ative air humidity, and Southern Oscillation Index) have been done
by Barrozo et al.17They concluded that correlations may reflect
enhanced fungal growth after increase in soil water storage in the
longer term and greater spore release with increase in absolute air
humidity in the short term.
Molecular epidemiology
tii are closely related sibling species that cause respiratory and
neurological disease in humans and animals. Within these two rec-
ognized species, phylogenetic analysis reveals cryptic species or
molecular types within the pathogenic Cryptococcus species com-
plex, corresponding to serotypes A (C. neoformans var. grubii; VNI,
VNII), D (C. neoformans var. neoformans; VNIV), AD (Hybrid; VNIII),
Page 4
G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
Mexico (n=69)
Spain (n=19)
Venezuela (n=20)
Brazil (n=66)
Argentina (n=57)
Colombia (n=62)
Guatemala (n=15)
Peru (n=13)
Chile (n=19)
Figure 1. Geographic distribution of the molecular types obtained from IberoAmerican Cryptococcus neoformans isolates by polymerase chain reaction fingerprinting and
URA5 gene restriction fragment length polymorphis analysis (total numbers studied per country given in parentheses).138Reproduced by permission.
bia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by
means of the M13 polymerase chain reaction-fingerprinting and
tion fragment length polymorphism (RFLP) analysis with HhaI and
Sau96I in a double digest.161The majority of the isolates (68.2%)
were VNI (C. neoformans var. grubii, serotype A), which agrees with
the fact of this variety being the cause of most human cryptococ-
cal infections worldwide, particularly in HIV-positive patients. Of
the remaining, 5.6% were VNII; 4.1% VNIII; 1.8%, VNIV; 3.5% VGI;
6.2% VGII; 9.1% VGIII, and 1.5% VGIV. Chile and Spain shared similar
molecular types, with a large number (15.8% and 42.1%, respec-
tively) of molecular type VNIII isolates (AD hybrids). VNIV serotype
D isolates were present only in Chile (26.3%). Patients with no
(fig. 1).161
A (91.1%) followed by serotypes B (8.4%) and C (0.5%) in clinical
samples, figures that moved to 44.2, 42.6 and 13.2%, respectively,
in environmental isolates. No serotype D or AD samples were iso-
of clinical serotype A and environmental serotype B isolates were
grouped into the molecular types VNI (98.1%) and VGII (100%),
respectively. Molecular type VGII was the predominant genotype
(77.7%) in both clinical and environmental Colombian C. gattii iso-
lates. This contrasts with previous reports in which VGII was only
found occasionally in tropical and subtropical regions.161
followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3%
each), and VNIII (< 1%).268Primary cryptococcosis caused by C.
gattii, molecular type VGII, prevailed in immunocompetent hosts,
mainly young people and children, in the North and Northeast
regions where C. gattii is endemic. On the other hand, in the Brazil-
ian Southern region, sporadic infections by C. gattii were recorded.
(21%), followed by VNII (5%), VGIII (4%), VGI and VNIV (3% each),
and VNIII (< 1%). Molecular type VGIV was not identified among
the Brazilian isolates.268
Out of 72 Mexican clinical isolates (PCR-fingerprinting with the
primer M13), 55 VNI, five VNII, three VNIII, one VNIV, two VGI, two
VGII, two VGIII and two VGIV isolates were reported.189The results
show that most cryptococcosis cases in Mexico are AIDS-related
In addition, this study revealed for the first time the presence of
genotypes VNIV and VGII among Mexican clinical isolates. Hence,
all genotypes that have been described for the Cryptococcus species
complex are found in Mexico, indicating a much wider geographic
distribution of genotypes than previously reported.
Using a different molecular approach, Díaz et al77employed
sequence analysis of the intergenic spacer regions, IGSI and IGSII,
the most rapidly evolving regions of the rDNA families. The
IGSI region displays the higher genetic variability, represented
by nucleotide base substitutions and the presence of long inser-
tions/deletions (indels). In contrast, the IGSII region exhibits less
heterogeneity and less extensive indels than the IGSI region.
Both intergenic spacers contain short, interspersed repeat motifs,
which can be related to length polymorphisms observed between
sequences. Phylogenetic analyses, undertaken in the IGSI, IGSII
and IGSI +5S rRNA+IGSII regions, revealed the presence of six
major phylogenetic lineages, some of which segregated into sub-
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
groups. The major lineages are represented by genotypes 1 (C.
and genotypes 3 to 6 represented by C. gattii, not always coinci-
dent with the molecular types found in the previously reviewed
H. capsulatum is a dimorphic fungus that has been recognized
as an important worldwide pathogen, agent of histoplasmosis. The
disease, which in some Latin American regions is a public health
threat, presents a wide diversity of clinical manifestations. Studies
on chromosomal band profiles of clinical isolates might shed light
on the role of fungal genetic diversity in the evolution of different
clinical forms of the disease. Using pulsed-field gel electrophoresis,
Canteros et al42analyzed intact chromosomes of 19 clinical iso-
lates of H. capsulatum isolated in Argentina, Mexico and Guatemala
and the laboratory reference strain G186B from Panama. Chromo-
somal banding patterns, grouped in 13 different electrokaryotypes,
ranged between 5 and 7 bands, 1.3 to 10 Mbp in size. Strain G186B
produced five bands of approximately 1.1, 2.8, 3.3, 5.4 and 9.7 Mbp.
Such chromosomal variability did not correlate with geographical
or clinical source. In spite of the apparently high chromosome-
length polymorphism, three clusters of identical patterns were
identified. The largest group, karyotype I, included only Argen-
tinean isolates of clinical origin, although this was not the only
karyotype in harbouring isolates from this country. All Mexican H.
capsulatum isolates were polymorphic among them.
Clinical observations in some Latin American countries indicate
that the lymphocutaneous form of sporothricosis is prevalent in
Mexico and Guatemala, whereas the fixed cutaneous form pre-
vails in Colombia. Mesa-Arango et al.160aimed to determine the
genotypic and phenotypic relatedness among Sporothrix schenckii
isolates in these countries. Clinical and environmental isolates of
S. schenckii were subjected to RAPD analysis-PCR with 10-mer
primers OPBG-01, OPBG-14, and OPBG-19. The 44 S. schenckii iso-
lates fell into four major groups by hierarchical cluster analysis.
Group I cluster together 25 out of 27 Mexican isolates, into two
isolates. Group II also split into two subgroups: IIa, Colombian iso-
lates, and IIb, Guatemalan isolates. Groups III and IV each had only
one clinical Mexican isolate. The low thermotolerance at 35 and 37
oC of the Colombian isolates could be associated with superficial
skin lesions in patients with fixed clinical forms of sporotrichosis,
the most frequent form of the disease in Colombia. Even though
the isolates were grouped by geographical origins, a high degree
of genotypic variability was observed among the isolates. Report-
ing a sporothrichosis epidemic in Rio de Janeiro, Brazil, Reis et al208
For this, the RAPD technique with three different primers and DNA
fingerprinting using the minisatellite derived from the wild-type
and 25 cat S. schenckii isolates into 5-10 genotypes. The RAPD pro-
files of epidemic S. schenckii isolates could be distinguished from
that of the United States-reference isolate, displaying 20% similar-
ity to each primer and 60% when amplified with the M13 primer.
the oral cavities (66%) of cats were identical to related human sam-
ples, suggesting that a common infection source for animals and
of S. schenckii.
By cladistic analysis of partial sequences of the calmodulin gene
using the maximum parsimony and neighbor-joining methods,
Madrid et al133determined that one out of 25 isolates from Mex-
ico (4%), one out of three isolates from Guatemala (33.3%), and two
bosa, while all other isolates belonged to S. schenckii sensu stricto,
this being the first record of S. globosa from Mexico, Central and
South America.
Cell biology
Metabolic and regulatory processes in growth and morphogenesis
Cell cycle and interaction between DNA replication, nuclei seg-
regation and budding in P. brasiliensis have been poorly studied.
Almeida et al3focused on the characteristics of the cell cycle pro-
file of P. brasiliensis yeast cells during batch culturing and under
the effects of benomyl, an antifungal drug known to promote a cell
cycle arrest in the G2/M phases of Saccharomyces cerevisiae. Their
results suggested that even though benomyl progressively blocks
nuclear division of P. brasiliensis yeast form, treated cells retained
their capacity for DNA replication.
ways to allow them to quickly adapt to a changing environment.
Most prominent among them are the mitogen-activated protein
kinase cascades. Some aspects of such complex systems are under
study in Latin America. The cAMP-dependent protein kinase (PKA)
from C. albicans is a tetramer composed of two catalytic subunits
and two type II regulatory subunits encoded by TPK1 and TPK2,
respectively, whose autophosphorylation site in Ser180 possibly
conforms a modulatory mechanism for C. albicans PKA activity in
vivo.287,288TPK1 is a positive regulator of the morphogenetic tran-
sition of C. albicans in the absence of the TPK2 gene.58The loss
of one catalytic isoform is not compensated by overexpression
of the other.245During Y-M transition, a sharp increase in TPK1
mRNA levels and in PKA-specific activity correlated with the onset
of germ-tube formation in strain tpk2?, reinforcing the idea that
Tpk1p is important for faster germ-tube appearance.
Bcy1p is a regulatory subunit of the PKA catalytic subunits TPK1
and TPK2.100BCY1-C. albicans yeast cells were used to generate a
double bcy1 tpk2 mutant,47with which it was proven that its con-
stitutive PKA activity was cAMP independent, indicating that the
cells harbored an unregulated phosphotransferase activity. Strains
with one BCY1 allele displayed pseudohyphae and true hyphae,
while hyphal morphology was almost exclusive in strains hav-
ing both BCY1 alleles, suggesting a tight regulation of PKA activity
for hyphal growth.100Further work101with mutants having het-
erozygous or homozygous deletions of TPK1 and/or TPK2 indicated
that tpk1?/tpk1? strains developed a lower tolerance to saline
gen storage, whereas wild-type and tpk2?/tpk2? mutants were
resistant to these stresses and accumulated higher levels of the
polysaccharide, indicating that both isoforms play different roles
in the stress response pathway and carbohydrate metabolism. In
like growth and opposes mycelial development.52
The Y-M transition in S. schenckii responds to protein kinase C
by Southern blot.8The latter has an ORF of 3942 nucleotides inter-
rupted by five introns, to encode a protein of 1194 amino acids and
132.84 kDa. pkcSs-2 is expressed at all intervals during the Y-M
transition.8Also in S. schenckii, a G?isubunit was found in a study
aimed to search the role of G proteins in signal transduction, the
first time such subunit was reported in a pathogenic fungus.76The
cDNA sequence revealed a 1059bp ORF encoding a 353 amino acid
G?isubunit of 41 kDa.
A homolog of the Pho85 cyclin-dependent kinase (Cdk) was
found in S. schenckii.75Pho85 has been identified as a regulator
of phosphate metabolism and modulator of the transcriptional
response to nutritional signals. The phoSs gene consists of 990bp,
contains one intron, and encodes a protein of 306 amino acids.
Expression of the phoSs gene decreased 30-fold during the Y-M
transition. The addition of extracellular calcium accelerated the
dimorphic transition and restored phoSs expression, suggesting
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
that PhoSs may participate in the control of the Y-M transition in S.
schenckii.75As an initial step to understand the PHO pathway in A.
fumigatus, de Gouvea et al.74characterized the PHO80 homologue,
PhoBPHO80and showed that the ?phoBPHO80mutant has a delayed
germ tube emergence; by phenotypic and phosphate uptake anal-
yses, the authors were able to establish a link between PhoBPHO80,
calcineurin and calcium metabolism. Several genes of the Pho
complex, namely, phoDPHO84, phoEPHO89, phoCPHO81, and vacuolar
transporter Vtc4 were more expressed both in the ?phoBPHO80
mutant background and under phosphate-limiting conditions of
0.1mM Pi. ?phoBPHO80and ?phoDPHO84mutant strains were fully
virulent in a murine low dose model for invasive aspergillosis.
The glyoxylate cycle, apparently involved in fungal pathogenic-
ity (for a review, see Dunn et al83), allows for the use of lipids
in the synthesis of glucose via acetate → citrate → isocitrate. Its
two initial steps are identical to those in the citric acid cycle. After
cleavage into succinate and glyoxylate and further condensation
with acetyl-CoA, malate is produced. Malate synthase is present in
P. brasiliensis; with a calculated 539 amino acids and a molecular
in the binding of fungal cells to the host, behaving as an anchorless
adhesion system.176
During the infective process, pathogenic fungi are subjected to
a significant environmental stress, including exposure to reactive
oxygen and nitrogen species produced by host cells. Mitochon-
dria are the main source of reactive oxygen species which need
to be controlled by detoxification mechanisms. Tudella et al269
analyzed an alternative oxidase and an uncoupling protein in the
respiratory chain of A. fumigatus. A functional respiratory chain
(complex I-V) was demonstrated: adenosine 5'-diphosphate (ADP)
induced an oligomycin-sensitive transition from resting to phos-
malate, glutamate, alpha-ketoglutarate, pyruvate, dihydroorotate,
succinate, N,N,N',N'-tetramethyl-p-phenylenediamine and exoge-
nous NADH. They were also able to demonstrate the presence of
an alternative NADH-ubiquinone oxidoreductase, an alternative
oxidase and an uncoupling protein in the respiratory chain of A.
fumigatus. Cloning and functional expression of the mitochondrial
alternative oxidase of A. fumigatus indicated that its gene (Afaox)
is 1173bp long, and encodes a 40 kDa protein.135In P. brasilien-
sis mitochondria, a complete (Complex I-V) functional respiratory
chain was also demonstrated.145An alternative NADH-ubiquinone
oxidoreductase, malate/NAD(+)-supported respiration, and alter-
the existence of alternative respiratory chain pathways in addi-
tion to Complex I in P. brasiliensis.145Similar results were found
in C. albicans mitochondria51,224and C. parapsilosis.162Because
such pathways are absent in animal cells, they may be excep-
mitochondrial genes coding for enzymes involved in the respira-
tory electron-transport chain, namely, proline oxidase, riboflavin
kinase, and cytochrome c oxidase, have been reported in the der-
matophyte Trichophyton rubrum.233
Flavoprotein monooxygenases constitute a family of enzymes
involved in a remarkably wide variety of oxidative reactions and
are, therefore, oxidoreductases; they are mainly related to anti-
oxidative stress in fungi and participate in several metabolic
pathways. One such protein has been identified in P. brasiliensis.140
It is the glycoprotein gp70, a concanavalin A-binding compo-
nent recognized by about 96% of sera from untreated patients
with PCM. Its gene encodes for a 79 kDa protein 718 aminoacids
long. An increased PbGP70 transcript accumulation is observed
under H2O2-induced oxidative stress, during fungal growth, and
in macrophage phagocyted/bound yeasts. In this way, GP70 may
work as a protector against oxidative stress and as elicitor of an
immune response.140Also related to oxidative stress are catalases,
whose main function is to prevent the oxidative damage triggered
by the reactive oxygen species of the host. Three catalases have
been reported in P. brasiliensis, two of which (CatA and PbCatC)
are monofunctional catalases and the third one (CatP), a cata-
lase peroxidase54; additionally, P. brasiliensis has both cytosolic
and peroxisomal catalase isoenzymes and a single cytochrome-c
peroxidase.70PbCatA manifested higher activity in the mycelial
phase, during M-Y transition or endogenous oxidative stress.
PbCatP showed higher activity in yeast cells since it is putatively
involved in the control of exogenous reactive oxygen species. In C.
glabrata, a high resistance to oxidative stress is mediated by a sin-
gle catalase, Cta1p, controlled by the transcription factors Yap1p,
Skn7p, Msn2p, and Msn4p.66C. dubliniensis, on the other hand,
mounts an adaptive response to stress that leads to an increased
survival against lethal doses of H2O2–like oxidants,266character-
ized by the induction of enzymes with known antioxidant function
(glucose-6-phosphate dehydrogenase, superoxide dismutase and
displaying higher susceptibility to their toxic effects.266
Enzymes whose encoding genes are differentially expressed
through themorphogenetic
Ornithine decarboxylase (ODC) is associated to the metabolism
of polyamines.210Early work indicated that in C. albicans, Mucor
rouxii, and Y. lipolytica, the activity of ODC was higher in the
mycelial phase; in P. brasiliensis, instead, it is the yeast phase
that shows a higher activity of the enzyme, either at the extreme
phases or through mycelial to yeast transition.230However,
PbrODC expression remained constant at all stages of the fungal
growth, a result that suggests a post-transcriptional regulation of
the PbrODC product.182C. immitis ODC, cloned in the pETCiODC
plasmid under control of T7lac promoter, was produced in trans-
formant strains Escherichia coli BL21(DE3), BL21(DE3)pLysS and
BLR(DE3) (? DE3 lysogen), and EWH319 (odc- null mutant).194E.
coli BL21(DE3)pLysS-pETCiODC expressed the highest specific ODC
activity, suggesting that this strain could be successfully used for
protein structure and drug testing studies.
Calcineurin is a Ca2+/calmodulin-dependent, serine/threonine-
specific phosphatase essential for adaptation to environmental
stress, growth, morphogenesis, and pathogenesis in many fungal
species. Calcineurin controls hyphal and yeast morphology, M-
Y dimorphism, growth, and Ca2+homeostasis in P. brasiliensis.40
In fungi calcineurin acts largely through regulating Crz1p-like
transcription factors. The A. fumigatus CRZ1 homologue CrzA was
characterized240; it is involved in mediation of cellular tolerance to
increased concentrations of calcium and manganese, also affecting
conidiation. Additionally, crzA-mutants suffer altered expression
of calcium transporter mRNAs under high concentrations of cal-
complemented and wild-type strains. The actual calcineurin A
gene (calA), coding for the catalytic subunit, is involved in hyphal
morphology related to apical extension and branching growth,
as shown by the defective and drastically decreased filamenta-
tion in calA-A. fumigatus mutants.136Such mutants also showed
an increased alternative oxidase (aoxA) mRNA accumulation and
activity. The authors also identified four transcription factors (zfpA,
htfA, nosA, and ctfA) that have increased mRNA expression in the
absence of calcineurin, suggesting a negative regulation by this
phosphatase. The deletion of the genes encoding these transcrip-
tion factors yielded disturbed mRNA accumulation of pmcA and
pmcB encoding calcium transporters. These deletion strains were
also less susceptible to itraconazole, caspofungin, and SDS.
TOR (target of rapamycin) is a pathway by which a regulation
is exerted on the translation of ribosomal proteins and, in yeast, of
ribosome biogenesis. In C. albicans, morphogenesis towards hyphal
process have beenreported.
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
of TOR, in the culture medium. Additionally, lithium suppressed
hyphal outgrowth in C. albicans in a way that also suggested inhi-
bition of the TOR pathway.144a,145
Proteinases occur naturally in all organisms, and are involved
in a multitude of physiological reactions. They are divided into
proteinases. Aspartyl- and serine-proteinases have been reported
in several fungal species by Latin American research groups. Of the
of P. brasiliensis was identified in the yeast cell wall. The expression
of putative genes CdSAP1, CdSAP2, CdSAP3, and CdSAP4 coding for
secreted aspartyl proteases of C. dubliniensis were reported.126,196
In addition, CdSAP7, 8, 9, and 10, orthologous genes of C. albi-
cans, were recognized in C. dubliniensis genome. The expression
of CdSAP1 and 2 was independent of the morphological stage of
C. dubliniensis.196CdSAP3 expression, instead, was related to the
infective process of keratinocytes. Expression of CdSAP4 predomi-
infection. These results suggest a role of C. dubliniensis Saps as vir-
ulence factors, similar to those from C. albicans.126Genome mining
and phylogenetic analyses revealed the presence of new mem-
bers of the Sap superfamily in C. tropicalis (8), C. guilliermondii (8),
C. parapsilosis (11) and Candida lusitaniae (3).196An extracellular
aspartyl-related proteolytic activity was also detected in mycelial
and conidial forms of F. pedrosoi. Pepstatin A was able to inhibit the
ing a possible participation of aspartyl peptidases in growth and
Exocellular serine-proteinases have been reported in P.
brasiliensis284and C. immitis.127In the latter, the mycelial 25 kDa
peptidase was able to degrade keratin while an additional 18 kDa
serine peptidase activity was evidenced solely when casein was
used as the substrate. In P. brasiliensis, Venancio et al274reported a
kexin-like gene (Pbkex2) codifying for a kexin protein that belongs
to the subtilase family of serine-proteinases. It is conformed by
an open reading frame (ORF) of 2622bp interrupted by one sin-
gle 93bp intron. The deduced protein sequence consists of 842
amino acid residues.264,274Also serine proteinases are the Lon
proteins, with roles in the maintenance of mitochondrial DNA
integrity and mitochondrial homeostasis. A LON gene homologue
from P. brasiliensis (PbLON) was identified by Barros and Puccia.16
PbLON ORF is within a 3,369-bp fragment interrupted by two
introns located in the 3' segment; an MDJ1-like gene was partially
5' untranslated region.19The authors propose that this chromo-
somal organization might be functionally relevant, since Mdj1p is
a type I DnaJ molecule located in the yeast mitochondrial matrix
and is essential for substrate degradation by Lon and other stress-
inducible ATP-dependent proteinases. An exocellular serine-thiol
proteinase (PbST) activity was reported by Matsuo et al146,147in
the yeast phase of P. brasiliensis. It was capable of cleaving pro-
teins associated with the basal membrane, such as human laminin
serine peptidase was identified in C. albicans that was active over
a broad pH range (5.0-7.2) and was able to hydrolyze some solu-
ble human serum proteins and extracellular matrix components.79
Conversely, when this isolate was grown in yeast carbon base
supplemented with bovine serum albumin, a secretory aspartyl
peptidase activity was measured, instead of metallo- and serine
peptidases, suggesting that distinct medium composition induces
different expression of released peptidases in C. albicans. Also in C.
A involved in the maturation of ?-factor mating pheromone. This
2793 pb gene is homozygotic and encodes for a predicted protein
of 930 amino acids with a molecular weight of 107 kDa. STE13ca
increases its levels of expression in conditions of nutritional stress
(proline as nitrogen source) and during formation of the germinal
Selective degradation of intracellular proteins in eukaryotic
cells is carried out by a 26S proteasome/polyubiquitin system,
in which polyubiquitin-labeled proteins are marked for destruc-
tion by the proteasome (for a review, see Sorais et al238).
The basic unit of the 26S proteasome is the 20S proteasome,
which in C. albicans yeast cells has a MW 640 kDa, distributed
within 14 polypeptides.88The enzyme shows chymotrypsin-like,
trypsin-like, and peptidylglutamyl-peptide-hydrolyzing activities.
The regulation of its activity may be mediated, in part, by phospho-
rylation, as suggested by experiments in vivo, using homologous
protein kinase CK2 as the substrate.89
Ecto-ATPases have been reported in F. pedrosoi59and C.
neoformans.116In the presence of 1mM EDTA, F. pedrosoi fungal
cells hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 84.6
+/- 11.3 nmol Pi/ h/ mg mycelial dry weight, while a value of
29.36+/-3.36nmol Pi/h/108cells was reported for C. neoformans.
MgCl2(0.05mM) was able to increase such activities 5 and 70
times, respectively. Based on their differential expression in the
different morphological stages of F. pedrosoi, a possible role in this
process was suggested. Since inhibition of ectophosphatase activ-
ity in cryptococci results in smaller rates of association of fungi
with animal epithelial cells, it was proposed that ectophosphatase
in C. neoformans may contribute to fungal colonization of the ani-
pedrosoi cell walls.118It was strongly inhibited by exogenous inor-
ganic phosphate (Pi); on the other hand, removal of Piresulted in
a 130-fold increase of ectophosphatase activity. Conidia with high
in adhesion to host cells.
carrionii, Cladophialophora bantiana and Exophiala jeanselmei have
urease, gelatinase and lipase activity.244Instead, only phospho-
lipase was detected in F. pedrosoi, a result that prompted the
this species from other agents of chromomycosis.244On the other
hand, keratinases (but not elastase, lipase or DNase) produced by
Microspoum canis have been proposed as a virulence factor275due
to the strong correlation between high keratinase activity and the
development of symptoms in samples isolated from symptomatic
or asymptomatic dogs and cats.
Exocellular enzymes depend on a secretory system to carry
them out of the cellular environment. Bernardo et al22studied the
pre-vacuolar branch of exocytosis in C. albicans, and were able to
identify structural homologs of several S. cerevisiae pre-vacuolar
secretory genes, including the late-Golgi vacuolar protein sorting
ing frame whose deduced protein product is 73.3% similar to S.
cerevisiae Vps1p and includes GTP-binding regions that are con-
served in members of the dynamin-like GTPase family of proteins.
VPS1-mutants lost their ability to secrete extracellular proteinases,
and were incapable of producing filaments. Both facts are related
an important element in the pathogenic process.
Genes involved in fungal cell wall synthesis
The fungal cell wall structure and its involvement in the
dimorphic process has been a constant subject of research. In P.
brasiliensis, several papers report the cloning, characterization and
synthase and the regulatory small GTPase Rho2,237the chitin
synthase multigene family180–183,223,226,229and ?-1,3-glucanosyl-
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
?-1,3-Glucan is a fungal cell wall polymer synthesized by the
multi-subunit enzyme ?-1,3-glucan synthase (FKS). The only FKS
gene in P. brasiliensis (FKS1) has an open reading frame of 5942bp,
interrupted by two putative introns, and a deduced sequence
of 1926 amino acids. P. brasiliensis Fks1p is a transmembrane
protein.199Activation of ?-1,3-glucan synthase in P. brasiliensis
requires the participation of the PbrRHO1 product as the GTPase
regulatory subunit.237
The ?-1,3-glucan synthase gene (PbrAGS1) presents six exons
accounting for a putative coding region of 7293bp, separated by
five introns.237It encodes a predicted protein of 2431 amino acids,
with a calculated mass of 274 kDa. It is expressed in the Y phase,
where the polysaccharide is solely found. Comparison of the lev-
els of expression of P. brasiliensis AGS1 and RHO2 in the M and
Y stages of the fungus shows a direct correlation, suggesting a
post-transcriptional regulation of P. brasiliensis AGS1, through the
product of RHO2.237Also in this family is Cdc42, a pivotal molecule
in establishing and maintaining polarized growth for diverse cell
types, as well as during pathogenesis of certain fungi. Almeida et
al2evaluated its role during cell growth and virulence of the yeast
form of P. brasiliensis and found that the expression of PbCDC42
in yeast cells promoted a decrease in cell size and more homoge-
nous cell growth, altering the typical polymorphism of wild-type
cells. Reduced expression levels also led to increased phagocyto-
sis and decreased virulence in a mouse model of infection. Hence,
Pbcdc42p seems to be an important protein during host-pathogen
interaction, with special relevance to the polymorphic nature and
cell size in the pathogenesis of P. brasiliensis.
The third important polysaccharide component of P. brasiliensis
cell wall is chitin. It serves functions in strengthening the fungal
cell wall and protection of the cell against lysis provoked by the
internal turgor pressure; it also participates in the connection of
capsular polysaccharides to the cryptococcal cell wall, forming sol-
uble complexes with glucuronoxylomannan (GXM).96Cultivation
of C. neoformans in the presence of an inhibitor of glucosamine
6-phosphate synthase resulted in altered expression of cell wall
chitin. These cells formed capsules that were loosely connected to
the cryptococcal wall and contained fibers with decreased diam-
eters and altered monosaccharide composition. GXM, the major
capsular component, is synthesized in cytoplasmic compartments
and transported to the extracellular space in vesicles. Cytoplas-
mic structures associated to vesicular compartments and reticular
membranes are in close proximity to the polysaccharide. GXM was
generally found in association with the membrane of intracellular
compartments and within different layers of the cell wall.190Anal-
ysis of extracellular fractions from cryptococcal supernatants by
transmission electron microscopy in combination with serologic,
chromatographic and spectroscopic methods revealed fractions
containing GXM and lipids. These results indicate an intimate asso-
ciation of GXM and lipids in both intracellular and extracellular
spaces consistent with polysaccharide synthesis and transport in
membrane-associated structures.
GXM is also produced by species of the Trichosporon genus, i.e.,
Trichosporum asahii.95Trichosporal and cryptococcal GXM share
antigenic reactivity, but Trichosporum polysaccharide has smaller
effective diameter and negative charge. GXM anchoring to the cell
of chitin-derived oligomers with the polysaccharide. GXM from T.
asahii supernatants are incorporated by acapsular mutants of C.
neoformans, which renders these cells more resistant to phago-
cytosis by mouse macrophages. Despite similarities in cell wall
anchoring, antigenic and antiphagocytic properties, trichosporal
and cryptococcal GXMs manifested major structural differences
that may affect polysaccharide assembly at the fungal surface.95
Chitin synthesis is controlled by a multigene family, some of
them redundant. Based on differences in regions of high sequence
conservation, chitin synthases have been organized according to
their amino acid sequences into two domains and seven classes.222
In P. brasiliensis six different chitin synthase genes have been
identified.180,181,263PbrCHS5 has a 5583 bp-long ORF, interrupted
by three introns of 82, 87 and 97bp. The deduced PbrCHS5 pro-
tein contains 1861 amino acids with a predicted molecular weight
of 206.9 kDa.181,183Two domains are identified, one towards
the N-terminal end of the protein (aa 16 to 786), with partial
identity to myosin motor-like domains, and a second one towards
the C-terminal end (aa 1221 to 1752) with homology to fun-
gal chitin synthases. PbrChs4, while being a protein as large as
PbrChs5, lacks sequences characteristic of myosin motors in its
N-terminal region.180,1835'UTR sequencing overlaps with a previ-
head-to-head configuration with CHS5, in a similar fashion as MDJ1
and LON,19mentioned in a previous section. P. brasiliensis CHS3 is
the only one to have a higher expression in the yeast phase and
at the end of the mycelium-yeast transition15; it contains a single
ORF 3817bp long with two introns (71 and 86bp) encoding a 1220
amino acid polypeptide with high similarity to other fungal chitin
chs2 was chosen by Matute et al151to study background selec-
tion at the locus in P. brasiliensis species complex. For this, the
DNA sequence for the chs2 locus was determined in 67 samples.
Of the 16 nucleotide substitutions located in the coding regions,
5 of them were synonymous and 11 non-synonymous. Because of
the very limited levels of polymorphism within each one of the P.
brasiliensis species and the low recombination levels observed in
this region, the observed data could be more likely explained by
the selective forces that affect loci over most of the chromosome,
but at a considerable distance from chs2.
In other fungal species, efforts have been addressed mainly to
cell wall-associated proteins and glycoproteins. In C. albicans, S.
cerevisiae and Y. lipolytica, cell wall proteins were either labeled
with biotin or radiolabeled with amino acids, and chased for a
period of time representing several generations. No significant
turnover took place during the chase period, and in fact radioactive
proteins were accumulated in the wall during the period, indicat-
ing that proteins bound to the cell wall are stable and that there
is no precursor-product relationship among those linked by non-
structure and synthesis of the cell wall of C. albicans display both
subtle and important differences with the wall of different sapro-
phytic fungi, of utmost importance for its pathogenic behavior (for
a review, see Ruiz-Herrera et al221).
Important cell wall proteins are adhesins that help in host-
pathogen interactions, inasmuch as adherence to target cells is
a prerequisite for fungal dissemination and systemic complica-
tions. Adherence to extracellular matrix (ECM) proteins has been
immobilized fibronectin122indicated that yeast cells and coni-
dia adhered equally to the glycoprotein, in a dose-dependent
manner; however, when the experiment was carried out with
soluble fibronectin, conidia displayed a very low binding capac-
ity compared to the yeast cells. This contradictory result may
be the consequence of tridimensional modification of the protein
structure, once this is subjected to an immobilization procedure
that leads to modifications on the exposure of adhesive domains,
as reported for other microorganisms.121S. schenckii binding to
fibronectin may be associated to the classical tripeptide arginine-
glycine-aspartic acid (RGD) adherence region of ECM molecules, a
conclusion derived from the fact that while S. schenckii binds to the
RGD-containing 120 kDa fibronectin fragment, inhibition assays
with RGDS and GRGDESP peptides did reduce adherence by 50%
to soluble fibronectin,121while no reduction was observed when
immobilized fibronectin was used.122Further research256aimed
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
to correlate S. schenckii virulence with protein pattern of cell wall
proteins and capacity to bind fibronectin indicated that no direct
cal isolates. The lowest virulence was found in an isolate recovered
from a patient with meningeal sporothrichosis. This isolate (IPEC
17943) exhibited the lowest capacity to interact with fibronectin,
and showed only one fibronectin-binding protein, a 67 kDa variant
of gp70 reported as a cell wall protein involved in fungal adherence
to dermal extracellular matrix.220The most virulent isolates (IPEC
15383 and 1099-8, from disseminated cutaneous and osteoven-
tricular, and subcutaneous sporothrichosis, respectively) showed a
higher adhesive capacity, and expressed at least four fibrinogen-
binding proteins (92, 55, 44, and 37 kDa) besides the 70 kDa band
characteristic of gp70.256
H. capsulatum yeast-cell binding to glycosylated surface
molecules of murine peritoneal or alveolar macrophages was
studiedusing attachmentinhibition
carbohydrate-treated yeastcells.81,254
the ?-anomer) and its derivatives were the most efficient sugar
inhibitors. These results suggested the presence of a lectin-like
component in H. capsulatum yeast cells and revealed involvement
of galactosylated surface molecules of murine macrophages as
specific-sugar (ligand) residues recognized by the fungal lectin.
H. capsulatum yeast cells are also able to bind to erythrocytes
irrespective of blood groups, an effect that could be inhibited not
only by galactose but also by galactose-containing disaccharides
and glycosaminoglycans, mainly chondroitin sulfate C, suggesting
a possible association of the inhibitory effect with the presence of
negative charges on the cell surface.254
In a histochemical study designed to evaluate the correlation
between the adherence of C. albicans and C. parapsilosis to human
buccal epithelial cells and the expression of fungal cell surface car-
bohydrates, Lima-Neto et al123found that adherence was higher in
glycoconjugates, suggesting that this monosaccharide may repre-
sent recognition molecules for interactions between the yeast and
the host. In C. glabrata, host-pathogen interaction in vitro depends
mainly on the adhesin Epa1, one of a large family of cell wall pro-
teins. Most of the EPA genes are located in subtelomeric regions,
where they are transcriptionally repressed by silencing. In order to
better characterize the transcriptional regulation of the EPA fam-
orthologues of known regulators of subtelomeric silencing (SIR2,
SIR3, SIR4, HDF1 (yKu70), HDF2 (yKu80), and RIF1) in S. cerevisiae.
They found that, whereas the SIR proteins are absolutely required
for silencing of the reporter genes and the native subtelomeric EPA
genes, the Rif1 and the Ku proteins regulate silencing at only a sub-
set of the analyzed telomeres. A cis element adjacent to the EPA3
locus can silence a reporter gene when placed at a distance of 31kb
from the telomere.219
The cell surface of C. albicans and other ascomycetous yeasts
is enriched in highly glycosylated mannoproteins that play roles
in the interaction with the host tissues. As with other bio-
logical systems, C. albicans protein glycosylation occurs mainly
through two distinct pathways, either O- or N-glycosylation.
Examples of the former are the enzymes dolichol phosphate
glucose synthase that catalyzes the transfer of sugar moieties
from either UDP-Glc to dolichol phosphate glucose,9the corre-
sponding mannose synthase, activated by cAMP-mediated protein
phosphorylation,10and the mannosyl transferase.11Example of
the latter is the N-glycosylation helped by ?-1,2-mannosidase
(MNS1), an enzyme involved in the hydrolysis of Man9GlcNAc2
and Man8GlcNAc2 oligosaccharides.166This goes by means of a
post-translational modification initiated in the endoplasmic retic-
ulum, where the Glc(3)Man(9)GlcNAc(2) N-glycan is processed by
with different
alpha-glucosidases I and II and alpha1,2-mannosidase to generate
Man(8)GlcNAc(2), enzymes codified by CWH41, ROT2, and MNS1,
respectively.166The N-oligosaccharide is then elaborated in the
Golgi to form N-glycans with highly branched outer chains rich in
mannose. Disruption of C. albicans CWH41, ROT2, and MNS1 leads
to mutants that tend to aggregate, have a lower content of cell
wall phosphomannan and other changes in cell wall composition,
and have a constitutively activated PKC-Mkc1 cell wall integrity
is converted into a cytosolic soluble enzyme with the help of the
Kex2 protease.165
The extraction of isolated cell walls from the yeast phase of S.
schenckii with SDS and separation of proteins by SDS-PAGE led
to the identification of a periodic acid-Schiff (PAS)-reacting 70
kDa glycoprotein (Gp70) that was purified by elution from elec-
trophoresis gels. The purified glycopeptide exhibited a pI of 4.1 and
about 5.7% of its molecular mass was contributed by N-linked gly-
distributed at the cell surface. Gp70 seems specific for S. schenckii
as no immunoreaction was observed in SDS-extracts from other
pathogenic and non-pathogenic fungi. Yeast cells of the fungus
serum reduced this process in a concentration-dependent manner,
suggesting Gp70 involvement in S. schenckii pathogenesis.220
cell wall in several species, where they contribute to the negative
residues are major anionogenic groups exposed on the P. brasilien-
sis surface, joined to galactose by means of ?-2,6- and ?-2,3- links.
Similar results were later reported when studying the cell-surface
expression of sialic acids in two isolates of C. albicans.236Sialic
acid reduces the binding of laminin and increases the binding of
fibronectin to S. schenckii yeast cells.121
Melanin has been proposed as a virulence factor in fungi.
Although not strictly a constituent element of the cell wall struc-
mesh, giving cells and colonies a characteristic brown to black pig-
mentation. Melanin is synthesized by laccase enzymes, a group of
multifunctional enzymes, in medium containing substrates such as
ical and 15 environmental isolates), belonging to serotypes A and
B, were analysed.198All strains showed laccase enzyme activity;
over half of the clinical strains of C. neoformans (56.2%) produced
the lowest melanin intensities, suggesting that melanin produc-
tion may not be the main virulence factor against host defence.
Furthermore, virulence could not be associated with the origin of
the sample, either clinical or environmental.
Fungal sphingolipids
pathogens have been characterized, and frequently involved
in host-pathogen interaction. Fungi also present unique glycol-
and/or disease establishment. The different biological roles for GSL
of different pathogens as infectivity factors and potential targets
for development of new therapeutic strategies have been reviewed
by Suzuki et al.248
Following studies on GSL in several dimorphic fungi, Taka-
hashi's group260–262analysed their structure, composition, and
dimorphic expression in S. schenckii. In lipids extracted from the
mycelial phase, a single cerebroside (Cer) component (glucosyl-
Cer) was observed, while in the yeast phase a galactosyl-Cer was
also detected. It is worth noting that glucosyl-Cer and its corre-
glycosphingolipids(GSL)from differenthuman
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
sponding synthase have been reported as a virulence factor in C.
neoformans.248The major long chain core in all three cerebrosides
was found to be (4E,8E)-9-methyl-4,8-sphingadienine, as reported
for the majority of fungi.261
Glycosylinositol phosphorylceramides
class of GSL that appear to be essential for fungal sur-
vival. In S. schenckii, GIPC structures were determined to be
(where Ins=myoinosytol, P=phosphodiester) in the mycelial
and the yeast phases.261An additional GIPC with the structure
Man?1→3Man?1→6GlcNH2?1→2Ins1-P-1Cer was reported in
both phases.262
Acidic GSL components were extracted from A. fumigatus and
identified as inositol phosphorylceramide and glycosylinositol
phosphorylceramides.259The structures of six major components
were elucidated as Ins-P-Cer, Man?1→3Man?1→2Ins1-P-1Cer,
→6]Man?1→2Ins1-P-1Cer, and Man?1→3Man?1→6GluN?1→
Similar glucosylceramide
ramide are present in A. nidulans, playing roles in germination
and hyphal growth, as demonstrated by their inhibition when
the fungus was treated with D-threo-1-phenyl-2-palmitoyl-3-
belonging to a family of compounds known to inhibit GlcCer syn-
thase in mammals.120Pseudallescheria boydii, a fungal pathogen
that causes disease in immunocompromised patients, also syn-
thesizes glucosylceramides as major neutral glycosphingolipids.
Ceramide monohexosides are detectable on the surface of mycelial
and pseudohyphal but not conidial forms of P. boydii, suggesting
a differential expression of glucosylceramides according with
the morphological phase. Addition of antiglucosylceramide anti-
bodies to cultures of C. albicans clearly inhibited the generation
of germ tubes, suggesting an involvement of ceramide mono-
hexosides in differentiation and infectivity.203In F. pedrosoi, the
main cerebroside species found in mycelia and conidial forms is
4,8-sphingadienine, while the major cerebroside species purified
from sclerotic cells carries an additional hydroxyl group, bound to
its long-chain base. The structural difference between cerebrosides
from mycelial and sclerotic cells was apparently not relevant for
their antigenicity, since they were both recognized at similar
levels by sera from individuals with chromoblastomycosis and a
monoclonal antibody to a conserved cerebroside structure.178
An interesting application of lipid biology to the clinics of
PCM comes from the work of Bertini et al.23By enzyme-linked
immunosorbent assay of sera from 31 PCM patients, these authors
analyzed immunoglobulin classes and isotypes of antibodies
directed to acidic glycosphingolipids (GSLs) and glucosylceramide
of P. brasiliensis. Only the GSL Pb-1 antigen, which presents the
carbohydrate structure Galf-?1-6(Man-?1-3)Man-?1, was reac-
tive with the PCM patient sera. The Galf residue is essential for
antibody reactivity, as shown by the lack of reactivity of Pb-2, the
The Pb-1 glycolipid from nontreated patients elicited a primary
immune response with immunoglobulin M (IgM) production and
subsequent switching to IgG1 production. The IgG1 titer increased
after the start of antifungal treatment, and general decreases in the
anti-Pb-1 antibody titers were observed after 5 months of treat-
ment. These results suggested that the Pb-1 antigen has potential
application as an elicitor of the host immune response in PCM
Sphingolipids and cholesterol, as important components of the
cell membrane, may be organized in membrane rafts that play
an essential role in different cellular functions, including host
cell-pathogen interaction. In P. brasiliensis, the involvement of
epithelial cell membrane rafts in the adhesion process of the
and Man?1→3Man?1→6Ins1-P-1Cer
pathogen and activation of cell signaling molecules was demon-
strated once the ganglioside GM1, a membrane raft marker, was
of fungal adhesion to host cells pre-treated with cholesterol-
extractor (methyl-beta-cyclodextrin) or cholesterol-binding (nys-
tatin) agents was additional proof of the interaction.36,151a
Lipid rafts may also be involved in the trafficking of polysaccha-
ride macromolecules from the cytoplasm to their final destination
in the outer cell wall, without breaking apart membranes.1,214,215
Recent reports indicate that extracellular vesicles, physiologically
secreted across the cell wall, help in the export process not only
of the major C. neoformans capsular polysaccharide glucuronoxy-
lomannan (average mass, 1.7 x 106to 7 x 106daltons),214but also
of a variety of virulence factors (e.g., glucosylceramides, laccase,
urease).214,215Additionally, 76 vesicle-located proteins were iden-
as vesicular proteins in mammalian exosomes.215Such vesicles
are built with bilayered membranes containing key fungal lipids,
such as GlcCer, and ergosterol, supporting the idea that they are
enriched in lipid rafts, and conforming a sophisticated trans-cell
wall vesicular transport secretory mechanism that is not avail-
able in prokaryotes; it may also indicate that extracellular vesicles
function as "virulence bags" that deliver a concentrated payload of
fungal products to host effector cells and tissues.211,214,215Analo-
gous findings have been reported in H. capsulatum,1suggesting a
general mechanism in fungi for the transport of virulence-related
macromolecules through vesicular secretion. Additionally, the fact
that similar vesicles have been found in species belonging in
ascomycetes (H. capsulatum) and basidiomycetes (C. neoformans)
gence of these branches 0.5-1.0 billion years ago.187
Transcriptome, genome, molecular taxonomy
Studies on the transcriptome of P. brasiliensis carried out by
Felipe et al.86and Goldman et al102have revealed expressed
such as cellular metabolism, information storage and processing,
cellular processes-cell division, posttranslational modifications,
morphogenetically-linked genes, among others. Molecular tech-
niques such as microarrays and substraction hybridization have
allowed the identification of genes involved in basic and cell
wall metabolism, sulfur metabolism, amino acid catabolism, sig-
nal transduction, growth and morphogenesis, protein synthesis,
genome structure, oxidative stress response, and development
genes that are preferentially expressed in the yeast phase,18,143,188
or differentially expressed in host-fungus interaction.13,65,252Since
which could represent novel exclusive conidia-yeast transition
Two fungal species, C. immitis and P. brasiliensis, both strongly
related to the Latin American region, have been the subject of
extraordinary advances in molecular taxonomy and phylogeny. C.
immitis, the etiologic agent of coccidioidomycosis, is endemic to
arid soils of the American continent, principally the lower Sonoran
life zone and desertic areas of Argentina and Venezuela.93Fisher et
al,93,94in a continental joint effort led by John Taylor at the Univer-
sity of California, Berkeley, were able to collect 161 clinical and two
environmental isolates, covering the known geographical range of
C. immitis. In them, allele distributions at the nine microsatellite
ing tree showed that isolates occur within one of two major clades,
the latter was renamed Coccidioides posadasii to honour Alejan-
dro Posadas, the Argentinean researcher who in 1892 reported the
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
fungus for the first time. It may represent a divergent, genetically
recombining monophyletic clade.93,94C. posadasii is the most fre-
quent species in the northern-central region of Mexico.49
P. brasiliensis is confined to the Latin American region.229This
fungus is considered clonal according to mycological criteria;
at the same time, it shows extensive genetic variability when
analyzed by molecular tools. RAPD analyses,37RFLP,179and partial
sequences of some genes112,164from a high number of P. brasilien-
sis isolates, revealed genetic variability and clusters correlated
with geography37,179or virulence.45,163Matute et al148analyzed P.
brasiliensis phylogenetically in search of cryptic species and found
that this fungus consists of at least three distinct, previously unrec-
ognized phylogenetic species: S1 (species 1 with 38 isolates of
assorted geographical origin), PS2 (phylogenetic species 2 with six
isolates, five Brazilian and one Venezuelan), and PS3 (phylogenetic
species 3, with 21 Colombian isolates). S1 and PS2 were sympatric
across their range, suggesting barriers to gene flow other than geo-
graphic isolation. Variations in virulence and gene expression of
antigenic proteins have been found between P. brasiliensis isolates
now known to belong to species S1 and PS2.112Despite their dif-
ferences, all three species are capable of inducing disease in both
humans and armadillos.45Matute et al150also developed a marker
system for DNA-based recognition of phylogenetic species S1 and
PS2 in P. brasiliensis, based on microsatellites. Searching for posi-
on the selection of 12 such genes involved in different cellular pro-
(p27 and gp43) have unknown functions. All other genes were clas-
sified in four different categories: metabolically related (fas2, his1),
cell wall related (fks, mnn5, ags1), heat shock proteins, detoxifi-
cation related (tsa1, sod1, hsp88) and signal transduction (cdc42,
cst20). Several replacement mutations in gp43 were under posi-
tive balancing selection. The other three genes (fks, cdc42 and p27)
showed very little variation among the P. brasiliensis lineages and
appeared to be under positive directional selection.
Following phylogenetic studies, Carrero et al44reported coding
the majority of the sequences used by Matute et al148and those
used in this study, grouped within two (S1 and PS3) of the three
clades proposed by these investigators. However, one P. brasilien-
sis isolate, Pb01, was placed at the base of, and quite distant from,
the three species reported by Matute et al,148clustering together
with strain IFM 54648, an atypical strain isolated from a patient
in the southern Brazilian region of Paraná.251This finding sug-
gested the possibility of more than three phylogenetic species in
P. brasiliensis.44Further work255gave strength to this hypothesis,
once the identification of 17 isolates, out of 88 samples, genotyp-
ically similar to strain Pb01, allowed their grouping as Pb01-like
isolates. They are considered a new phylogenetic species distinct
from the S1, PS2 and PS3 clades previously reported by Matute et
al,148since it is strongly supported by all independent and con-
catenated genealogies, with highly significant values of posterior
probability (1.0) and bootstrap agreement (100%). The speciation
event that defined this new phylogenetic group is sympatric rela-
tive to S1 and PS2. The two separate groups that include S1, PS2,
PS3 on one side and Pb01-like on the other, were highly divergent
(fig. 2).255Based on molecular phylogenetic data, distinctive mor-
phological characters and a long period of genetic isolation (> 30
be considered a new phylogenetic species, and we proposed the
binomial name Paracoccidioides lutzii,255whose specific descriptor
means to honour Adolpho Lutz, the Brazilian researcher who first
reported P. brasiliensis in 1908.
Electrophoretic karyotypes of 12 clinical and environmental P.
brasiliensis isolates from different geographic areas indicated the
P. lutzii
218 717 189
351 61 54 397
769 133 206
P. brasiliensis
Figure 2. Bayesian unrooted phylogram showing the relationship between the iso-
lates from the three phylogenetic species S1, PS2, PS3 and the isolates from the
"Pb01-like" cluster. Eight concatenated loci, comprising 3,565 nucleotides, from
dataset 1 (fks-exon2, fks-exon3, chs2-exon1, chs2-exon2-4, gp43-promoter-exon1,
gp43-exon2, arf and ?-tubulin). This is a consensus unrooted tree highlighting the
site analysed [modified from 218]. Reproduced by permission.
possible existence of haploid and diploid (or aneuploid) isolates
of the fungus.85Further studies by flow cytometry and comparison
uninucleated yeast cell in 10 P. brasiliensis isolates. The analysis of
intra-individual variability of the highly polymorphic P. brasiliensis
gp43 gene164indicated that only one allele was present; therefore,
all isolates presented a haploid, or at least aneuploid, DNA content;
no association was detected between genome size/ploidy and the
clinical-epidemiological features of the isolates.3
One extraordinary step forward in the field has been the recent
public release of the genome of three P. brasiliensis strains, among
them, the above mentioned Pb01 isolate, in an effort led by the
Broad Institute, MIT, Boston, that included all Latin American
laboratories involved in molecular biological research of the fun-
gus (Brazil, Colombia and Venezuela), under the Paracoccidioides
Comparative Genome Analysis Project. Data can be found at
brasiliensis.2/MultiHome.html. Preliminary data-mining analyses
indicate that the Pb01 strain does have important differences with
the other two isolates, Pb18 and Pb03, particularly with regards
to the genome size (32.94, 29.06 and 29.95 Mb, respectively)
and number of genes (9132, 7875 and 8741 genes, respectively)
[manuscript in preparation], a result that provides additional
arguments in favour of the proposed classification of Pb01-like
isolates as P. lutzii.
Despite their telomeric (sexual) stages or mating system being
unknown, C. immitis, C. posadasii, P. brasiliensis and P. lutzii have
been classified by molecular criteria as belonging to the phylum
Ascomycota, Order Onygenales.229Recent work265aimed to deter-
mine the presence of the mating type locus in 71 P. brasiliensis
isolates from various sources. Two heterothallic groups (MAT1-1
was confirmed, indicating the existence of a basal gene expression.
The distribution of two mating type loci in the studied population
suggested that sexual reproduction might occur in P. brasiliensis.
This finding points towards the possibility of applying a more pre-
cise definition of the concept of biological species to P. brasiliensis.
Beginning with the first reported human case of Jorge Lobo's
disease, its etiologic agent, Lacazia loboi, has been at the center
of a taxonomic dispute. The fungus was described as Loboa loboi
but subsequent morphological, serological and molecular studies
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
phylogenetic analysis using 20 Lacazia loboi isolates (as the species
ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-
ribosylation factor, and gp43 and compared them to those from 17
P. brasiliensis strains that represented the known variation in this
species,44,148,277and outgroup taxa in the Onygenales (Ajellomyces
and Coccidioides species). Nucleotide variation among strains of L.
loboi was minor but numerous nucleotide mismatches and multi-
ple gaps were found for these gene regions among members in the
Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred
using neighbor-joining, maximum parsimony and Bayesian analy-
ses depicted L. loboi as a well-supported, monophyletic group that
was sister to the Paracoccidioides clade. The authors concluded
that L. loboi should be maintained as a taxon independent from
Paracoccidioides within the Ajellomycetaceae.276
Innate immunity effector mechanisms in mycoses
One major development in the field of immunology is the
acknowledgment that innate immunity, although not being
highly specific, is able to efficiently detect microbial infections
through the recognition of pathogen-associated molecular pat-
terns (PAMPs) by specialized pattern recognition receptors (PRRs);
oligomerization domain-like receptors (NOD), dectin C, and oth-
ers. These evolutionarily conserved structures are mainly present
in monocytes, macrophages, dendritic cells, T and B lymphocytes,
mediating the recognition of microbial pathogens and the subse-
quent inflammatory and immune responses.156In fungi, dectin-1,
mannose receptor, TLR4, TLR2, and galectin-3 that recognize ˇ-
(1,3)-glucans, mannans, mannoproteins, phospholipomannan and
ˇ-mannosides, respectively, have been identified (reviewed by
Gow et al110).
The simultaneous activation of multiple PRRs by a fungal
pathogen directs the immune system to mount an ample and
effective specific immune response against the fungus. Thus, the
immunity but also in orchestrating the adaptive immunity devel-
oped in sequence. Many fungal wall components are recognized by
of TLR, is dispensable for resistance to P. brasiliensis and that TLR2,
TLR4 and dectin-1 do not play a significant role in the recognition
of P. brasiliensis yeast cells. However, further research90implicated
in Brazil has extensively studied the role of MyD88 and also of
TLR2 and TLR4 in experimental paracoccidioidomycosis.130They
demonstrated that TLR2 deficiency resulted in the development
of milder infection, decreased nitric oxide synthesis and increased
IL-17.38Dectin-1, CD18, and TLR2 receptors are also involved in
the lipid body formation induced by the cell wall ?-glucan of H.
capsulatum, a phenomenon linked to leukotriene B4 generation.239
Studies with C. albicans showed that the interaction of this fungal
agent with intrahepatic lymphocytes resulted in the up-regulation
of TLR-2 expression in this cell population.209The involvement
of TLR4 in the recognition of S. schenckii was suggested by stud-
ies that compared TLR4 deficient and sufficient mice and which
showed that both pro-inflammatory and anti-inflammatory medi-
ators were reduced in the TLR4-deficient group.232Therefore, the
involvement of TLR4 in S. schenckii recognition by the host was first
described by Latin American authors.
A critical point at the initial stage of antifungal defense is
the production of chemotactic factors (cytokines, chemokines and
leukotriens) at the site of the infection, for the effective recruit-
ment of phagocytes (neutrophils, monocytes and macrophages),
dendritic and natural killer cells. For instance, the cytokine IFN-?
modulates the chemokine production and leukocyte recruitment
to the lungs of P. brasiliensis-infected mice.246On the other hand,
H. capsulatum induced generation of high levels of MIP-1-?, and of
low levels of eotaxin and its ?-glucan cell wall component induced
a little MIP-1-? but considerably higher concentrations of eotaxin,
suggesting that chemokines and leukotrienes may play key roles in
the inflammatory cell influx to H. capsulatum infection.154
The production of pro-and anti-inflammatory cytokines and
influences not only the inflammatory response that is mounted
shortly after the infection but also enhances or impairs the sub-
sequent development of an effective protective immune response,
being therefore decisive to the outcome of the subsequent disease
developed. For this reason, the effect of each fungal agent on the
production of different types of these mediators was the object of
study of many Latin American authors.
Some of these results strongly suggest that an imbalance in the
production of pro-inflammatory and anti-inflammatory cytokines
maybeassociatedwiththepathogenesisofsomemycoses.Perac ¸oli
et al197have characterized the cytokines produced by monocytes
from PCM patients and were able to demonstrate that endoge-
nous levels of TNF-?, IL-1?, IL-6, IL-8, IL-10 and TGF-?, detected
in monocytes from patients, were significantly higher than those
produced by healthy controls. IL-18, a recently described cytokine,
important in the regulation of both innate and acquired immune
response, was also studied in PCM. The results showed that IL-18
sis than their wild type controls.193Mamoni and Blotta138showed
that gene expresión, kinetics of cytokines and chemokines distin-
guishes P. brasiliensis infection from disease, as deduced from the
earlier and higher levels of TNF-?, IFN-? and chemokines mRNAs
in PCM-infected patients as compared with patients suffering from
the juvenile form of the disease. Preferential induction of pro-
inflammatory cytokines was also demonstrated at the onset of
experimental P. brasiliensis infection.107Patients with chromoblas-
tomycosis produce high levels of IL-10 and low levels of IFN-?,
resulting in the development of a somehow impaired immune
Phagocytes, (neutrophils and macrophages), which constitute
the first cell population to confront the fungus after infection,
have an important role, not only in the events directly related
with fungal lysis but also with the activation of acquired immu-
nity. Macrophages are activated by different mechanisms in order
to perform their function with increased efficiency. Receptors for
complement system components, mannans and ?-glucan (like
dectin 1) activate different pathways to make sure that fungi are
et al.115in relation with complement and mannose receptors.
Employing congenic murine bone-marrow-derived macrophage
lines infected with P. brasiliensis conidia, the authors suggested the
vating effect on the antifungal activity of these cells by cytokines,
mainly IFN-?.
Human phagocytes were also studied in this mycosis. Mono-
cytes obtained from PCM patients, were preactivated with
recombinant IFN-? and evaluated for their fungicidal activity
against P. brasiliensis. Cells from healthy subjects failed to present
such activity while those from PCM patients showed significant
fungicidal activity against virulent Pb18; in contrast, both patient
and control cells were significantly fungicidal against avirulent
Pb265.39Some factors can also inhibit phagocytosis: monocytes
sis, suggesting a role of prostaglandin E2 (PGE2) in the inhibitory
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
process. Human monocytes challenged with the fungus produced
high PGE2 levels, which in turn repressed the fungicidal activ-
ity by reducing H2O2and TNF-? production.31Some fungi are
able to secrete products that alter the fungicidal properties of
macrophages. Indeed, a lipid component of the S. schenckii cell wall
was shown to inhibit phagocytosis by macrophages.43
Apoptosis induction may lead to the impairment of some
cells that play essential roles in the establishment of protective
immunity against fungal infections. Peritoneal macrophages co-
incubated with C. albicans strain CR1 in vitro show early signs of
apoptosis, but evolve to necrosis after 2h. At the same time, an
increase in IL-10 production is observed. Treatment of CR1 with
pepstatin (a proteinase inhibitor) prevented the process of apop-
tosis and significantly reduced IL-10 production, suggesting that
the increased production of this cytokyne was caused by processes
occurring during the initial phase of infection, such as apoptosis,
necrosis and uptake of death cells.99The liver constitutes the first
barrier in the control of hematogenous dissemination of C. albi-
cans of intestinal origin. Renna et al.209studied the involvement
of apoptosis and pro-apoptotic signals in the hepatic injury dur-
ing the acute phase of C. albicans infection and concluded that
in the scenario of early liver injury, the recruited intrahepatic
lymphocytes and the modulated expression of TNF-?, Fas-L and
TLR-2 molecules could act coordinately in delivering death sig-
Neutrophils are also deeply affected by cytokines, which can
enhance or impair their phagocyitic activity. IFN-?, as well as the
more recently studied cytokine IL-15 facilitate P. brasiliensis killing
by human neutrophils, in contrast to the effect of IL-10.12,64,253
Using an experimental model of candidosis, it was shown that
neutrophils, at a first phase and later, macrophages, are involved
in clearing an experimental infection by C. albicans.131F. pedrosoi
experimental inoculation elicited marked neutrophils migration
to the inflammatory site followed by microbicidal activity, par-
ticularly against hyphae, suggesting that host resistance to this
fungus is primarily mediated by neutrophils. As a high number of
destroyed conidia was found intracellularly in macrophages, the
further participation of these cells is suggested.132
The destruction of phagocytes through induction of apoptosis
has been described in this review as a mechanism of fungal escape
from immune response. H. capsulatum is a facultative intracellular
parasite, found in neutrophils and mononuclear cells, suggesting
The work of Medeiros et al153shows that H. capsulatum-infected
leukocytes presented less apoptosis than controls, suggesting that
this fungus induces an antiapoptotic state on neutrophils and
monocytes. This phenomenon may represent an extraordinary
escape mechanism, by delaying cell death and allowing H. capsula-
tum to survive inside phagocytes.
Macrophages and dendritic cells (DCs) are antigen-presenting
cells with a fundamental role in connecting the innate and the
acquired immune responses. DCs in particular have been recently
recognized as initiators and modulators of immune responses and
tion site and transformed into mature DCs. They recognize fungal
cells by their TLR receptors, and are induced to produce proin-
flammatory cytokines such as TNF-?, IL-1, IL-6, IL-8 and IL-15.
Cytokines, as well as co-stimulatory molecules are needed to ren-
der lymphocytes fully activated and so trigger acquired immune
The role of DCs is essential in subcutaneous mycoses, such
as chromoblastomycosis, because its causative agent, the fungus
F. pedrosoi and DCs have plentiful opportunity to interact at the
onset of the infection. DCs from patients exhibited an up-regulated
expression of human leucocyte antigen D-related (HLA-DR) and of
co-stimulatory molecules (CD86). In the presence of conidia, the
expression of HLA-DR and CD86 was up-regulated by DCs from
studies in dermatophytosis, it was shown that T. rubrum-infected
macrophages have down-regulated expression of co-stimulatory
molecules (CD80 and CD54) and that the ingested conidia grow
and differentiate into hyphae inside macrophages, leading to
rupture of the cell membrane, suggesting the existence of
mechanisms that evade or suppress protective immunity.41The
expression of co-stimulatory molecules was also studied on T-
cells and monocytes of active PCM patients and healthy individuals
cured of past PCM. CD28 expression, critical for optimal T-cell acti-
vation, was comparable between patients and controls, whereas
CD152, PD-1 and ICOS, which preferentially deliver negative sig-
from patients. CD80 and CD86 were equally expressed on mono-
The recognition of fungal cells by the innate immunity leads to
that have as a consequence the formation of an inflammatory envi-
ronment for the recognition of the pathogen, the mounting of the
cellular or humoral immune response.
Mechanisms of humoral immunity–Role of specific antibodies in
During the last decade it has been demonstrated that, although
not as determinant as cellular immunity, humoral immune
protection against fungal infections, provided that protective anti-
bodies be present in adequate concentration.46,205Although the
protective role of specific antibodies against P. brasiliensis is still
controversial, the contribution of Taborda's group was essential for
the understanding of the humoral arm of anti-fungal immunity.
In fact, Buissa-Filho et al35studied the effects of monoclonal anti-
bodies to the major P. brasiliensis antigen (gp43) using in vitro and
in vivo infection models. As reported by the authors, the passive
administration of monoclonal antibodies against gp43 before and
after infection led to a reduced fungal burden and decreased pul-
by these cells.
In a series of studies on mycotic patients, several Latin Ameri-
can authors detected patterns of preferential isotypes production
according to the clinical manifestations of each mycosis. For
instance, Biselli et al27found that 100% of patients with the severe
acute form of PCM produced high levels of anti-gp43 antibod-
ies of IgE, an isotype which is preferentially synthesized in a Th2
cytokines environment, whereas only 27% of the patients with the
chronic form of the disease produced this isotype at high levels.
These results support the notion that the relatively more severe
impairment of cellular immunity in the acute form of PCM is prob-
ably related to a Th2 pattern of immune response.
Patients with other mycoses were also the object of such stud-
ies. The IgG, IgM, and IgA immunoglobulins in sera of patients with
sporotrichosis before antifungal treatment and also from patients
with sporotrichosis during itraconazole treatment were quanti-
fied. More than 95% of patients had detectable IgA antibodies, and
more than 85% had IgM and IgG antibodies before treatment. The
number of patients with IgG antibodies increased to 91% during
treatment. Conversely, significantly fewer samples from treated
patients were positive for IgM (71%) and IgA (89%). Overall, 78% of
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this percentage dropped to 62.9% in patients that were receiving
The majority o f substances that elicit good immune responses
antigens. There are, however, some carbohydrate antigens that can
be recognized as native molecules by B lymphocytes; these are the
T-independent antigens. The fungus C. neoformans has a polysac-
charide capsule composed of glucuronoxylomannan (GXM), which
is such an antigen. Parra et al195quantified the production of IgG
moderately resistant (Balb/c), highly resistant (CBA/j) and suscep-
tible (C57BL/6) mouse strains. Early production of IgG1, described
as protector antibodies, coincided with a decrease of the number
of C. neoformans colony forming units in the lungs.
Some fungi are able to produce and secrete products that
interfere with components of the immune response, constituting,
eventually, fungal virulence factors. Antigenic preparations from S.
schenckii yielded proteases that are were able to cleave different
subclasses of human IgG.68
The knowledge that antibodies against fungi could be protec-
tive led to the development of a very original approach by a group
of Latin American and Italian researchers. They tested the in vitro
fungicidal and the in vivo therapeutic activity of an engineered
synthetic decapeptide was derived from the sequence of a recom-
binant anti-idiotypic antibody, that represents the internal image
of a Pichia anomala killer toxin. This compound markedly reduced
the fungal load in organs (liver, lung, spleen) of mice infected with
P. brasiliensis, opening a new field in the induction of protective
immunity against fungi.267
Mechanisms of cellular immunity–Role of lymphocytes and
cytokines in mycoses
There is evidence of efficient activation of cell-mediated immu-
nity after exposure to fungi. Lymphocytes from healthy subjects
show strong proliferative responses against fungal antigens, pro-
tissue response against fungal invasion is a granulomatous inflam-
matory response, characteristic of cellular immunity. Resistance
against fungi is based on a triple response, i.e., induction of a
strong cellular immune response mediated by T helper lympho-
cytes with CD4 phenotype, production of cytokines and action of
effector phagocytes (fig. 3).
The necessity of a strong, functional cellular immune response
is illustrated by chronic mucocutaneous candidosis patients, who
clear cells from these patients produced lower levels of IFN-? and
IL-2 than controls in response to Candida antigens, but did not pro-
duce higher levels of IL-4 and IL-10, suggesting that, even though
Th1 cytokines are decreased, the Th2 response is not increased in
this severe form of candidosis.36In cryptococcosis, however, the
presence of capsular polysaccharidic components induce a domi-
nant Th2 pattern, with high levels of IL-4 and IL-10 production and
undetectable inflammatory cytokines, such as TNF-? and IFN-?,
constituting a powerful virulence factor.4
Corbellini et al61studied the delayed-type hypersensitivity
response developed against F. pedrosoi exoantigens in 16 male
guinea pigs, all but one showing positive response 48h after inoc-
ulation, results that show that a specific T cell response, develops
after exposure to chromoblastomycosis. The combined effects of
both CD4+and CD8+T lymphocytes and Th1 and Th2 cytokines are
required for the induction of resistance to various fungi. Chiarella
et al57demonstrated in a murine model that CD8+T cells were
the major elements involved in the control of P. brasiliensis loads,
whereas CD4+T cells were responsible for delayed type hypersen-
sitivity responses and antibody production. Teixeira de Souza et
showed that absence of CD4(+) cells induces a more severe disease
in chromoblastomycosis.
Th1 lymphocytes produce predominantly IFN-? and elicit
phagocyte activation leading to respiratory burst. In contrast, Th2
the synthesis of antibodies, resulting in susceptibility to fungal
infections and allergic reactions. Some fungi subvert the Th1/Th2
dicothomy in their favor. C. neoformans GXM profoundly alters the
phenomena. It was shown that it suppresses lymphoproliferation
in response to either concanavalin A or heat-killed C. neoformans,
modulates cytokine production, determining high production of
IL-10, and low secretion of IL-2, IFN-? and TNF-? and also
triggers macrophage apoptosis through NO generation.55,56The
capsular polysaccharides, galactoxylomannan (GalXM) and glu-
curonoxylomannan (GXM) induced different cytokines profiles in
macrophages. GalXM induced production of TNF-?, NO and iNOS
expression, while GXM induced predominantly TGF-? secretion,
but both induced macrophages apoptosis mediated by Fas/FasL
interaction. All these phenomena constitute mechanisms by which
capsular polysaccharides from C. neoformans might compromise
host immune responses.278
High levels of IL-10 and TNF-? in the sera of chronic PCM
patients have been reported.97Marques-Mello et al.144associ-
ated IL-4 and IL-5 production with a Th2 immune response to P.
brasiliensis infection. Romano et al. showed that PCM-related Th1
immunosuppression was associated with down-modulation of the
IL-12 pathway, and that patients cured from PCM may not fully
recover their immune responsiveness.218The group of Blotta char-
acterized the immune response of PCM patients suffering from
responses, preferential production of antibody isotype IgG1 in uni-
focal, milder cases, or IgE and IgG4 in multifocal severe cases; in
the juvenile form, instead, Th2 response dominates, with produc-
tion of IgA, IgE and IgG4 antibodies.139They also associated IL-18
and TNF receptor 2 with the severity of the disease.62In experi-
mental models, it was demonstrated that the absence of functional
tects mice against disseminated infection but enhances pulmonary
inflammation,12suggesting that resistance to P. brasiliensis infec-
tion correlates with preferential Th1 immune response.117
This may also be the case in other mycoses. Severe forms
of chromoblastomycosis are characterized by the production of
high levels of IL-10 and TNF-?, associated to low levels of IFN-
? and lymphocyte proliferation, in contrast with the mild cases,
in which low levels of IL-10, high levels of IFN-? and good lym-
phocyte proliferation were observed.152It has been described
that immune modulation with recombinant IL-12 or anti-IL-10
can restore the antigen-specific Th1-type immune response in
chromoblastomycosis patients by up-regulating HLA-DR and co
stimulatory molecules in monocytes.242Zaga-Clavellina et al283
reported that C. albicans induced differential synthesis and secre-
tion of IL-1?, IL-6 and prostaglandin-E.
Da Silva et al69demonstrated that Langerhans cells were able
to phagocytose F. pedrosoi conidia but not sclerotic cells, inhibiting
hyphae formation. The development of local immune responses at
the site of the granulomatous lesions and the interaction of fungi
with extracellular matrix components and cytokines was studied
in P. brasiliensis106,185,186and S. schenckii models.91
The role of nitric oxide (NO) in the fungicidal effect
of macrophages has been a recurrent subject of research.
Macrophages represent the major cell defense against fungi; when
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G. San-Blas, E. Burger / Rev Iberoam Micol. 2011;28(1):1–25
Figure 3. Histological analysis of granulomatous lesions in B10.A mice, susceptible to P. brasiliensis. The organ was collected 15 days after IP infection with the highly
virulent Pb18 isolate. This figure illustrates the close interaction of different cell populations with the immune response in the course of experimental infection. A.Compact
granulomatous lesion with presence of yeast cells, inflammatory cellular influx and extracellular matrix components. Magnification x200; HE dye. B.Marked influx of
phagocytic cells of different lineages, some showing ingested P. brasiliensis cells. Magnification ×400; HE dye. C. Concentric collagen fibers circumscribing the granuloma and
eventually containing fungal dissemination. Magnification x200; Sirius red dye observed under Polarized light. D. Marked deposition of thick collagen fibers delimiting the
lesion. Magnification x400; Sirius red dye. E. P. brasiliensis at the center of the lesion. Magnification x200; Groccot dye. F. P. brasiliensis with preserved or altered morphology.
Magnification x400; Groccot dye. Extracellular matrix components (blue arrows), neutrophils (green arrows), multinuclear giant cells (red arrows), fungi with preserved
(violet arrows) or altered (brown arrows) morphology. Figure authors: RFS Molina and E. Burger. Original figure for this manuscript.
mechanism via the enhanced production of NO. However, the role
studies of various fungi, albeit numerous, are mostly inconclusive.
One example is the persistence of Cryptococcus in the central
nervous system in spite of marked local expression of mRNAs of
nitric oxide synthase (iNOS) and stimulatory cytokines: increased
levels of transcripts corresponding to IL-1, TNF-? and iNOS were
detected as early as day 1 post infection, with TNF-? rising by
approximately 30-fold and iNOS increasing by approximately 5-
fold by day 7.134
In PCM an inverse correlation between NO concentration and
et al184showed that NO has an important role in granuloma mod-
ulation, by controlling OPN and MMP production, as well as by
inducing loose granulomas formation and fungal dissemination,
resulting, at later phases, in PCM progression, thus confirming ear-
lier results by Nascimento et al,175who described the dual role of
this gas in experimental PCM. Moreira et al,168in turn, were able
to show an association of IFN-? or TNF-?-activated macrophages
cells, an effect reversed with the addition of inhibitors. The role
of nitric oxide synthase (iNOS) in PCM has also been studied by
Livonesi et al124who suggest that iNOS is a resistance factor in
PCM by controlling fungal proliferation, influencing cytokines pro-
duction, and appeasing the development of a high inflammatory
response and concurrent necrosis.
NO was found to be fungicidal against S. schenckii. Mice defec-
tive in the production of reactive nitrogen intermediates (iNOS-/-
mutants) or wild-type mice treated with an iNOS inhibitor were
used to investigate the role of endogenous NO during systemic
sporotrichosis. The results suggest that although NO was an essen-
tial mediator to the in vitro killing of S. schenckii by macrophages,
the activation of NO system in vivo contributes to the immuno-
suppression and cytokine balance during early phases of infection
with this fungus.87Using in vivo and in vitro models of chromoblas-
tomycosis, Bocca et al30demonstrated that, during the infection,
F. pedrosoi peritoneal macrophages show an increased phagocytic
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