Phenotypic variation and intracellular parasitism by Histoplasma capsulatum

The success of Histoplasma capsulatum as an intracellular pathogen depends completely on successful conversion of the saprophytic mycelial (mold) form of this fungus to a parasitic yeast form. It is therefore not surprising that yeast phase-specific genes and gene products are proving to be important for survival and proliferation of H. capsulatum within macrophages. In this study, we have focused on the role and regulation of two yeast-specific characteristics: α-(1,3)-glucan, a cell wall polysaccharide modulated by cell-density (quorum) sensing, and a secreted calcium-binding protein (CBP) that is essential for pathogenicity.

Defining responses to therapy and study outcomes in clinical trials of invasive fungal diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer consensus criteria.

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

Attempts at a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy

Chemotherapy is the basis of treatment of paracoccidioidomycosis in its various forms. Depending on the Paracoccidioides brasiliensis virulence, the status of host immunity, the degree of tissue involvement and fungal dissemination, treatment can be extended for long periods with an alarming frequency of relapses. Association of chemotherapy with a vaccine to boost the cellular immune response seemed a relevant project not only to reduce the time of treatment but also to prevent relapses and improve the prognosis of anergic cases. The candidate immunogen is the gp43 major diagnostic antigen of P. brasiliensis and more specifically its derived peptide P10, carrying the CD4(+) T-cell epitope. Both gp43 and P10 protected Balb/c mice against intratracheal infections with virulent P. brasiliensis strain. P10 as single peptide or in a multiple-antigen-peptide (MAP) tetravalent construction was protective without adjuvant either by preimmunization and intratracheal challenge or as a therapeutic agent in mice with installed infection. P10 showed additive protective effects in drug-treated mice stimulating a Th-1 type immune response with high IFN-gamma and IL-12. P10 and few other peptides in the gp43 were selected by Tepitope algorithm and actually shown to promiscuously bind several prominent HLA-DR molecules suggesting that a peptide vaccine could be devised for a genetically heterogenous population. P10 was protective in animals turned anergic, was effective in a DNA minigene vaccine, and increased the protection by monoclonal antibodies in Balb/c mice. DNA vaccines and peptide vaccines are promising therapeutic tools to be explored in the control of systemic mycoses.

Malassezia spp. in acoustic meatus of bats (Molossus molossus) of the Amazon region, Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, `` Rondonia ``, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N=24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (""ghosts"") can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.

Pathogenesis II: Fungal responses to host responses: interaction of host cells with fungi

Most of our knowledge concerning the virulence determinants of pathogenic fungi comes from the infected host, mainly from animal models and more recently from in vitro studies with cell cultures. The fungi usually present intra- and/or extracellular host-parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules. Among living organisms, this has been characterized as a cohabitation event, where the fungus is able to recognize specific host tissues acting as an attractant, creating stable conditions for its survival. Several fungi pathogenic for humans and animals have evolved special strategies to deliver elements to their cellular targets that may be relevant to their pathogenicity. Most of these pathogens express surface factors that mediate binding to host cells either directly or indirectly, in the latter case binding to host adhesion components such as extracellular matrix (ECM) proteins, which act as 'interlinking' molecules. The entry of the pathogen into the host cell is initiated by fungal adherence to the cell surface, which generates an uptake signal that may induce its cytoplasmic internalization. Once this is accomplished, some fungi are able to alter the host cytoskeletal architecture, as manifested by a rearrangement of microtubule and microfilament proteins, and this can also induce epithelial host cells to become apoptotic. It is possible that fungal pathogens induce modulation of different host cell pathways in order to evade host defences and to foster their own proliferation. For a number of pathogens, the ability to bind ECM glycoproteins, the capability of internalization and the induction of apoptosis are considered important factors in virulence. Furthermore, specific recognition between fungal parasites and their host cell targets may be mediated by the interaction of carbohydrate-binding proteins, e.g., lectins on the surface of one type of cell, probably a parasite, that combine with complementary sugars on the surface of host-cell. These interactions supply precise models to study putative adhesins and receptor-containing molecules in the context of the fungus-host interface. The recognition of the host molecules by fungi such as Aspergillus fumigatus, Paracoccidioides brasiliensis and Histoplasma capsulatum, and their molecular mechanisms of adhesion and invasion, are reviewed in this paper.

Mycoses associated with AIDS in the Third World

Despite advances in diagnosis and treatment, the epidemiological status of the human immunodeficiency virus (HIV) infection is far from under control in most of the developing world. Sub-Saharan Africa, Southeast Asia and India show increased rates of new infections. In Latin America and the Caribbean there were 1.6 million estimated cases of HIV-infected patients at the end of 1997. Fungal diseases have been one of the most relevant diagnoses in relation to the acquired immunodeficiency syndrome (AIDS), Infections due to Candida species and Cryptococcus neoformans var, neoformans are common worldwide. Histoplasma capsulatum, Coccidioides immitis and Penicillium marneffei are important causes of disease in endemic areas. Infection due to Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis are uncommon even where they are endemic. Phaeohyphomycetes, hyalohyphomycetes and zygomycetes are still rare as a cause of disease among AIDS patients, However, agents pertaining to these groups, such as Aspergillus spp., have an increasing incidence. Superficial mycoses due to dermatophytes have special features from epidemiological, clinical and therapeutic points of view.

Histoplasmosis presenting as cellulitis 18 years after renal transplantation

A 49-year-old renal transplant patient, under an 18-year course of immunosuppressive therapy with prednisone and azathioprine and, more recently, prednisone plus mycophenolate sodium, developed a cutaneous-subcutaneous infection caused by Histoplasma capsulatum. The clinical presentation consisted of a slowly enlarging, erythematous and infiltrative 25 cm plaque in the major axis on the arm. There was no involvement of the lungs or any other organ. Cure was obtained with itraconazole treatment after 12 months. Histoplasmosis is an uncommon opportunistic infection among solid organ transplanted patients with incidence of 0% to 2.1% observed in a large number of cases. This report describes an atypical cutaneous clinical presentation of a potentially fatal disease in immunosuppressed patients.

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12 000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or internal proteins are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

PRP8 intein in Ajellomycetaceae family pathogens: Sequence analysis, splicing evaluation and homing endonuclease activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Defense reactions of Dermatobia hominis (Diptera : Cuterebridae) larval hemocytes

The defense reactions against biological (Histoplasma capsulatum and Escherichia coli) and non-biological materials (China ink and nylon thread) were tested in vivo in third instar larvae of Dermatobia hominis. The cellular defense performed by larval hemocytes was observed under electron microscopy. China ink particles were phagocytosed by granular cells 5 h after injection. E. coli cells were internalized by granular cells as early as 5 min after injection and totally cleared 180 min post-injection, when many hemocytes appeared disintegrated and others in process of recovering. H. capsulatum yeasts provoked, 24 h after being injected, the beginning of nodule formation. Nylon thread was encapsulated 24 h after the introduction into the hemocoel. Our results suggest that granular cells were the phagocytic cells and also the responsible for the triggering of nodule and capsule formation. In the presence of yeasts cells and nylon thread, they released their granules that chemotactically attracted the plasmatocytes that on their turn, flattened to surround and isolate the foreign material.

Virulence Factors IN Fungi of Systemic Mycoses

Fatores de virulência em fungos de micoses sistêmicaFungos patogênicos causadores de micoses sistêmicas possuem vários fatores que permitem seu crescimento nas condições adversas oferecidas pelo hospedeiro, propiciando o estabelecimento da relação parasitária e contribuindo no processo de doença. Esses fatores são conhecidos como fatores de virulência auxiliando no desenvolvimento da infecção e interferindo com a patogênese das micoses. O presente trabalho avalia os fatores de virulência em fungos patogênicos como Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum e Paracoccidioides brasiliensis, em relação à termotolerância, dimorfismo, componentes da parede celular ou cápsula, bem como a produção de enzimas. Os fatores de virulência auxiliam na aderência, colonização, disseminação e habilidade do fungo para resistir a ambientes hostis e escapar dos mecanismos da resposta imune do hospedeiro.Tanto os fatores de virulência apresentados por diferentes fungos, como os mecanismos de defesa oferecidos pelo hospedeiro requerem ação e interação de processos complexos, cujo conhecimento permitirá a melhor compreensão da patogenia das micoses sistêmicas.

Immunosuppression in paracoccidioidomycosis: T cell hyporesponsiveness to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral immune response

To assess human cellular immune response to paracoccidioidomycosis (PCM), lymphocyte proliferative responses to purified antigens from Paracoccidioides brasiliensis were determined in healthy persons previously infected by the fungus (positive donors), in healthy noninfected persons (controls), and in PCM patients. Affinity-purified gp70 and gp43, the two major antigens in humoral immune responses, were used, Both induced lymphocyte proliferation (gp43 species-specific) in positive donors but not in controls; healthy persons previously infected by Histoplasma capsulatum reacted to gp70 and not to gp43, A similar cross-reactivity in antibody response to gp70 was previously reported; however, antibody response to gp43 has been considered specific, Lymphocytes from PCM patients, who, unlike positive donors, have high levels of anti-gp43 and anti-gp70 antibodies, proliferated poorly with gp70 and gp43 but better with other stimuli, This dichotomy between humoral and cellular antigen-specific responses suggests a Th2 immune response in PCM, which may be related to failure to control the infection.

EARLY ACTIVATION OF SPLENIC MACROPHAGES BY TUMOR-NECROSIS-FACTOR-ALPHA IS IMPORTANT IN DETERMINING THE OUTCOME OF EXPERIMENTAL HISTOPLASMOSIS IN MICE

Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally. The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells. Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice. Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection. Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion. It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.