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1.  The impact of antifungals on toll-like receptors 
Fungi are increasingly recognized as major pathogens in immunocompromised individuals. With the increase in the number of fungal infections each year and the development of resistance to current therapy, new approaches to treatment including stimulation of the immune response in addition to concurrent pharmacotherapy is ongoing. The most common invasive fungal infections are caused by Candida spp., Aspergillus spp., and Cryptococcus spp. Amphotericin B (AmB) has remained the cornerstone of therapy against many fulminant fungal infections but its use is limited by its multitude of side effects. Echinocandins are a newer class of antifungal drugs with activity against Candida spp. and Aspergillus spp. and constitutes an alternative to AmB due to superior patient tolerability and fewer side effects. Due to their oral delivery, azoles continue to be heavily used for simple and complex diseases, such as fluconazole for candidal vaginitis and voriconazole for aspergillosis. The objective of this paper is to present current knowledge regarding the multiple interactions between the broad spectrum antifungals and the innate immune response, primarily focusing on the toll-like receptors.
PMCID: PMC3954077  PMID: 24672516
antifungals; amphotericin B; echinocandins; caspofungin; voriconazole; toll-like receptors
2.  Galleria mellonella as a model host to study Paracoccidioides lutzii and Histoplasma capsulatum 
Virulence  2013;4(2):139-146.
Non-mammalian models have been used to investigate fungal virulence. In this work we have explored the use of Galleria mellonella as an infection model for the pathogenic dimorphic fungi Histoplasma capsulatum and Paracoccidioides lutzii. In mammalian models these fungi cause similar infections, and disease outcomes are influenced by the quantity of the infective inocula. We describe a similar aspect in a G. mellonella model and characterize the pathogenesis features in this system. Infection with P. lutzii or H. capsulatum, in all inoculum used, killed larvae at 25 and 37°C. However, there was a lack of correlation between the number of yeast cells used for infection and the time to larvae death, which may indicate that the fungi induce protective responses in a dynamic manner as the lowest concentrations of fungi induced the most rapid death. For both fungi, the degree of larvae melanization was directly proportional to the inocula size, and this effect was visibly more apparent at 37°C. Histological evaluation of the larvae showed a correlation between the inoculum and granuloma-like formation. Our results suggest that G. mellonella is a potentially useful model to study virulence of dimorphic fungi.
PMCID: PMC3654612  PMID: 23302787
Galleria mellonella; Paracoccidioides lutzii; Histoplasma capsulatum; infection model; fungal pathogenesis
3.  Mathematical Modeling Predicts Enhanced Growth of X-Ray Irradiated Pigmented Fungi 
PLoS ONE  2014;9(1):e85561.
Ionizing radiation is known for its cytotoxic and mutagenic properties. However, recent evidence suggests that chronic sub-lethal irradiation stimulates the growth of melanin-pigmented (melanized) fungi, supporting the hypothesis that interactions between melanin and ionizing photons generate energy useful for fungal growth, and/or regulate growth-promoting genes. There are no quantitative models of how fungal proliferation is affected by ionizing photon energy, dose rate, and presence versus absence of melanin on the same genetic background. Here we present such a model, which we test using experimental data on melanin-modulated radiation-induced proliferation enhancement in the fungus Cryptococcus neoformans, exposed to two different peak energies (150 and 320 kVp) over a wide range of X-ray dose rates. Our analysis demonstrates that radiation-induced proliferation enhancement in C. neoformans behaves as a binary “on/off” phenomenon, which is triggered by dose rates <0.002 mGy/h, and stays in the “on” position. A competing dose rate-dependent growth inhibition becomes apparent at dose rates >5000 mGy/h. Proliferation enhancement of irradiated cells compared with unirradiated controls occurs at both X-ray peak energies, but its magnitude is modulated by X-ray peak energy and cell melanization. At dose rates <5000 mGy/h, both melanized and non-melanized cells exposed to 150 kVp X-rays, and non-melanized cells exposed to 320 kVp X-rays, all exhibit the same proliferation enhancement: on average, chronic irradiation stimulates each founder cell to produce 100 (95% CI: 83, 116) extra descendants over 48 hours. Interactions between melanin and 320 kVp X-rays result in a significant (2-tailed p-value = 4.8×10−5) additional increase in the number of radiation-induced descendants per founder cell: by 55 (95% CI: 29, 81). These results show that both melanin-dependent and melanin-independent mechanisms are involved in radiation-induced fungal growth enhancement, and implicate direct and/or indirect interactions of melanin with high energy ionizing photons as an important pro-proliferative factor.
PMCID: PMC3893251  PMID: 24454887
4.  Methamphetamine Enhances Cryptococcus neoformans Pulmonary Infection and Dissemination to the Brain 
mBio  2013;4(4):e00400-13.
Methamphetamine (METH) is a major addictive drug of abuse in the United States and worldwide, and its use is linked to HIV acquisition. The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis in patients with AIDS. In addition to functioning as a central nervous system stimulant, METH has diverse effects on host immunity. Using a systemic mouse model of infection and in vitro assays in order to critically assess the impact of METH on C. neoformans pathogenesis, we demonstrate that METH stimulates fungal adhesion, glucuronoxylomannan (GXM) release, and biofilm formation in the lungs. Interestingly, structural analysis of the capsular polysaccharide of METH-exposed cryptococci revealed that METH alters the carbohydrate composition of this virulence factor, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Additionally, we show that METH promotes C. neoformans dissemination from the respiratory tract into the brain parenchyma. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.
Methamphetamine (METH) is a major health threat to our society, as it adversely changes people’s behavior, as well as increases the risk for the acquisition of diverse infectious diseases, particularly those that enter through the respiratory tract or skin. This report investigates the effects of METH use on pulmonary infection by the AIDS-related fungus Cryptococcus neoformans. This drug of abuse stimulates colonization and biofilm formation in the lungs, followed by dissemination of the fungus to the central nervous system. Notably, C. neoformans modifies its capsular polysaccharide after METH exposure, highlighting the fungus’s ability to adapt to environmental stimuli, a possible explanation for its pathogenesis. The findings may translate into new knowledge and development of therapeutic and public health strategies to deal with the devastating complications of METH abuse.
PMCID: PMC3735193  PMID: 23900172
5.  Characterization of Virulence Properties in the C. parapsilosis Sensu Lato Species 
PLoS ONE  2013;8(7):e68704.
The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12 C. metapsilosis and 18 C. orthopsilosis isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the C. metapsilosis strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to C. orthopsilosis and C. metapsilosis isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and C. metapsilosis strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of Galleriamellonella larvae. The mortality rate of G. mellonella larvae infected with C. metapsilosis isolates was significantly lower than those infected with C. parapsilosis sensu stricto or C. orthopsilosis strains. Taken together, our findings demonstrate that C. metapsilosis is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions.
PMCID: PMC3706360  PMID: 23874732
6.  The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur 
PLoS ONE  2013;8(6):e63764.
Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.
PMCID: PMC3676409  PMID: 23762233
7.  Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine 
Applied and Environmental Microbiology  2012;78(24):8623-8630.
Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.
PMCID: PMC3502921  PMID: 23042177
8.  A role for vesicular transport of macromolecules across cell walls in fungal pathogenesis 
In our recent work, we have shown that fungal species from different phyla produce extracellular vesicles. The vesicles are heterogeneous and morphologically similar to mammalian exosomes, with intact bilayered membranes. Proteomic analyses reveal that the vesicles contain a broad array of macromolecules, many of which are associated with fungal virulence. Further, the biological import of the extracellular fungal vesicles is supported by their presence during murine cryptococcosis and the immunoreactivity of convalescent serum from patients with Cryptococcus neoformans or Histoplasma capsulatum vesicle protein extracts.
In contrast to most eukaryotic cells, fungi have complex cell walls, that could in theory provide a significant barrier to the secretion of large molecules. The discovery of trans-cell wall vesicular transport in fungi provides a solution to the problem of extracellular transport of macromolecules. Identifying similar vesicles in ascomycetes and basidiomycetes suggest that the shuttle system is ancient, predating the divergence of these branches 0.5–1.0 billion years ago. Importantly, the discovery of this trans-cell wall vesicular transport system also poses new, interesting questions for future investigations.
PMCID: PMC2629580  PMID: 19169363
Histoplasma capsulatum; Cryptococcus neoformans; vesicles; pathogenesis; virulence; cell transport
9.  A role for vesicular transport of macromolecules across cell walls in fungal pathogenesis 
In our recent work, we have shown that fungal species from different phyla produce extracellular vesicles. The vesicles are heterogeneous and morphologically similar to mammalian exosomes, with intact bilayered membranes. Proteomic analyses reveal that the vesicles contain a broad array of macromolecules, many of which are associated with fungal virulence. Further, the biological import of the extracellular fungal vesicles is supported by their presence during murine cryptococcosis and the immunoreactivity of convalescent serum from patients with Cryptococcus neoformans or Histoplasma capsulatum vesicle protein extracts.
In contrast to most eukaryotic cells, fungi have complex cell walls, that could in theory provide a significant barrier to the secretion of large molecules. The discovery of trans-cell wall vesicular transport in fungi provides a solution to the problem of extracellular transport of macromolecules. Identifying similar vesicles in ascomycetes and basidiomycetes suggest that the shuttle system is ancient, predating the divergence of these branches 0.5–1.0 billion years ago. Importantly, the discovery of this trans-cell wall vesicular transport system also poses new, interesting questions for future investigations.
PMCID: PMC2629580  PMID: 19169363
Histoplasma capsulatum; Cryptococcus neoformans; vesicles; pathogenesis; virulence; cell transport
10.  Macrophage Autophagy in Immunity to Cryptococcus neoformans and Candida albicans 
Infection and Immunity  2012;80(9):3065-3076.
Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans, two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans. In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans, depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans, increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans, as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.
PMCID: PMC3418760  PMID: 22710871
The role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic.
PMCID: PMC3564053  PMID: 22411314
12.  Vaccines and immunotherapy against fungi: the new frontier 
PMCID: PMC3554959  PMID: 23355835
13.  Nitric oxide nanoparticles 
Virulence  2012;3(1):62-67.
Nitric oxide (NO) is a critical component of host defense against invading pathogens; however, its therapeutic utility is limited due to a lack of practical delivery systems. Recently, a NO-releasing nanoparticulate platform (NO-np) was shown to have in vitro broad-spectrum antimicrobial activity and in vivo pre-clinical efficacy in a dermal abscess model. To extend these findings, both topical (TP) and intralesional (IL) NO-np administration was evaluated in a MRSA intramuscular murine abscess model and compared with vancomycin. All treatment arms accelerated abscess clearance clinically, histologically, and by microbiological assays on both days 4 and 7 following infection. However, abscesses treated with NO-np via either route demonstrated a more substantial, statistically significant decrease in bacterial survival based on colony forming unit assays and histologically revealed less inflammatory cell infiltration and preserved muscular architecture. These data suggest that the NO-np may be an effective addition to our armament for deep soft tissue infections.
PMCID: PMC3337151  PMID: 22286699
MRSA; nanotechnology; nitric oxide; pyomyositis
14.  Inhibition of Candida parapsilosis Fatty Acid Synthase (Fas2) Induces Mitochondrial Cell Death in Serum 
PLoS Pathogens  2012;8(8):e1002879.
We have recently observed that a fatty acid auxotrophic mutant (fatty acid synthase, Fas2Δ/Δ) of the emerging human pathogenic yeast Candida parapsilosis dies after incubation in various media including serum. In the present study we describe the mechanism for cell death induced by serum and glucose containing media. We show that Fas2Δ/Δ yeast cells are profoundly susceptible to glucose leading us to propose that yeast cells lacking fatty acids exhibit uncontrolled metabolism in response to glucose. We demonstrate that incubation of Fas2Δ/Δ yeast cells with serum leads to cell death, and this process can be prevented with inhibition of protein or DNA synthesis, indicating that newly synthesized cellular components are detrimental to the mutant cells. Furthermore, we have found that cell death is mediated by mitochondria. Suppression of electron transport enzymes using inhibitors such as cyanide or azide prevents ROS overproduction and Fas2Δ/Δ yeast cell death. Additionally, deletion of mitochondrial DNA, which encodes several subunits for enzymes of the electron transport chain, significantly reduces serum-induced Fas2Δ/Δ yeast cell death. Therefore, our results show that serum and glucose media induce Fas2Δ/Δ yeast cell death by triggering unbalanced metabolism, which is regulated by mitochondria. To our knowledge, this is the first study to critically define a link between cytosolic fatty acid synthesis and mitochondrial function in response to serum stress in C. parapsilosis.
Author Summary
Candida parapsilosis is a human opportunistic pathogen associated with significant morbidity and mortality, especially in immunocompromised individuals such as premature, low-birthweight neonates. Our prior studies have indicated that C. parapsilosis effectively utilizes fatty acids/lipids for growth and virulence. We now show that inhibition of the fatty acid synthase (Fas2) results in a hypersensitivity to serum, indicating that yeast cell survival and replication in serum medium or in vivo is dependent on Fas2. Serum hypersensitivity of Fas2-inhibited yeast cells is due to mitochondrial mediated dysregulation of metabolism. Thus, we conclude that Fas2 is candidate antifungal target to combat disseminated fungal infections.
PMCID: PMC3431346  PMID: 22952445
15.  Melanogenesis in dermatophyte species in vitro and during infection 
Microbiology  2011;157(Pt 8):2348-2356.
Dermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.
PMCID: PMC3167886  PMID: 21565930
16.  Nitric Oxide Releasing Nanoparticles for Treatment of Candida Albicans Burn Infections 
Candida albicans is a leading fungal cause of burn infections in hospital settings, and sepsis is one of the principle causes of death after a severe burn. The prevalence of invasive candidiasis in burn cases varies widely, but it accounts for 3–23% of severe infection with a mortality rate ranging from 14 to 70%. Therefore, it is imperative that we develop innovative therapeutics to which this fungus is unlikely to evolve resistance, thus curtailing the associated morbidity and mortality and ultimately improving our capacity to treat these infections. An inexpensive and stable nitric oxide (NO)-releasing nanoparticle (NO-np) platform has been recently developed. NO is known to have direct antifungal activity, modulate host immune responses and significantly regulate wound healing. In this study, we hypothesized that NO-np would be an effective therapy in the treatment of C. albicans burn infections. Using a murine burn model, NO-np demonstrated antifungal activity against C. albicans in vivo, most likely by arresting its growth and morphogenesis as demonstrated in vitro. NO-np demonstrated effective antimicrobial activity against yeast and filamentous forms of the fungus. Moreover, we showed that NO-np significantly accelerated the rate of wound healing in cutaneous burn infections when compared to controls. The histological evaluation of the affected tissue revealed that NO-np treatment modified leukocyte infiltration, minimized the fungal burden, and reduced collagen degradation, thus providing potential mechanisms for the therapeutics’ biological activity. Together, these data suggest that NO-np have the potential to serve as a novel topical antifungal which can be used for the treatment of cutaneous burn infections and wounds.
PMCID: PMC3370663  PMID: 22701111
nitric oxide; nanoparticles; burn healing; collagen; Candida albicans
17.  The potential of nitric oxide releasing therapies as antimicrobial agents 
Virulence  2012;3(3):271-279.
Nitric oxide (NO) is a short-lived, diatomic, lipophilic gas that plays an integral role in defending against pathogens. Among its many functions are involvement in immune cell signaling and in the biochemical reactions by which immune cells defend against bacteria, fungi, viruses and parasites. NO signaling directs a broad spectrum of processes, including the differentiation, proliferation, and apoptosis of immune cells. When secreted by activated immune cells, NO diffuses across cellular membranes and exacts nitrosative and oxidative damage on invading pathogens. These observations led to the development of NO delivery systems that can harness the antimicrobial properties of this evanescent gas. The innate microbicidal properties of NO, as well as the antimicrobial activity of the various NO delivery systems, are reviewed.
PMCID: PMC3442839  PMID: 22546899
antibiotic; antimicrobial; nanotechnology; nitric oxide
18.  Detection of Antibodies against Paracoccidioides brasiliensis Melanin in In Vitro and In Vivo Studies during Infection ▿ 
Clinical and Vaccine Immunology : CVI  2011;18(10):1680-1688.
Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.
PMCID: PMC3187017  PMID: 21813659
19.  Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis 
Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.
Author Summary
Paracoccidioidomycosis (PCM) is the predominant systemic mycosis in Latin America causing half of the total deaths among systemic fungal infectious diseases in Brazil. Chemotherapy is the standard treatment, but the long time required, severe cases of immunosuppression and frequent relapses indicate that additional methods should be introduced such as immunotherapy combined with antifungal drugs. Previously, the protective activity of P10, a peptide derived from the major diagnostic antigen gp43, was demonstrated, alone or combined with chemotherapy. P10 elicited a vigorous IFN-γ mediated Th-1 immune response. Presently, the reduction of fungal load, and even sterilization, was attempted using a specific DNA vaccine encoding P10. Plasmid pcDNA3 expression vector with P10 insert was tested as a vaccine in intratracheally infected BALB/c and B10.A mice. Our results showed that vaccination with pP10 induced a significant reduction of the fungal burden in the lung. Co-vaccination of pP10 with a plasmid encoding mouse IL-12 proved to be even more effective in the elimination of the fungus with virtual sterilization in a long term infection and treatment assay system. The data suggest that immunization with these plasmids, without the need of an adjuvant, could be used in the prevention and treatment of PCM in human patients.
PMCID: PMC3289603  PMID: 22389734
20.  Antibody Therapy for Histoplasmosis 
The endemic human pathogenic fungus Histoplasma capsulatum is a major fungal pathogen with a broad variety of clinical presentations, ranging from mild, focal pulmonary disease to life-threatening systemic infections. Although azoles, such as itraconazole and voriconazole, and amphotericin B have significant activity against H. capsulatum, about 1 in 10 patients hospitalized due to histoplasmosis die. Hence, new approaches for managing disease are being sought. Over the past 10 years, studies have demonstrated that monoclonal antibodies (mAbs) can modify the pathogenesis of histoplasmosis. Disease has been shown to be impacted by mAbs targeting either fungal cell surface proteins or host co-stimulatory molecules. This review will detail our current knowledge regarding the impact of antibody therapy on histoplasmosis.
PMCID: PMC3270318  PMID: 22347215
Histoplasma capsulatum; histoplasmosis; antibody; histone 2B; heat shock protein 60; M antigen; co-stimulation
21.  Radioimmunotherapy of Fungal Diseases: The Therapeutic Potential of Cytocidal Radiation Delivered by Antibody Targeting Fungal Cell Surface Antigens 
Radioimmunotherapy is the targeted delivery of cytocidal radiation to cells via specific antibody. Although mature for the treatment of cancer, RIT of infectious diseases is in pre-clinical development. However, as there is an obvious and urgent need for novel approaches to treat infectious diseases, RIT can provide us with a powerful approach to combat serious diseases, including invasive fungal infections. For example, RIT has proven more effective than standard amphotericin B for the treatment of experimental cryptococcosis. This review will discuss the concepts of RIT, its applications for infectious diseases, and the strides made to date to bring RIT of infectious diseases to fruition. Finally, we will discuss the potential of PAN-FUNGAL RIT, the targeting of conserved fungal cell surface antigens by RIT, as a treatment modality for fungi prior to the formal microbiological identification of the specific pathogen. In sum, RIT provides a mechanism for the targeted killing of drug susceptible or resistant fungi irrespective of the host immune status and may dramatically reduce the length of therapy currently required for many invasive fungal diseases.
PMCID: PMC3257868  PMID: 22275913
Histoplasma capsulatum; Cryptococcus neoformans; Candida albicans; antibody; heat shock protein 60; beta-glucan; melanin
22.  Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major 
Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H4B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H4B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H4B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah− null mutant and an episomal complemented overexpressing derivative (pah−/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah− parasites showed reactivity with an antibody to melanin when grown with L-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah− showed an increased sensitivity to Tyr deprivation, while the pah−/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah− showed no alterations in H4B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.
PMCID: PMC2987234  PMID: 20887755
trypanosomatid protozoa; tetrahydrobiopterin; aromatic amino acid hydroxylase; amino acid metabolism; virulence
23.  Surface Architecture of Histoplasma Capsulatum 
The dimorphic fungal pathogen Histoplasma capsulatum is the most frequent cause of clinically significant fungal pneumonia in humans. H. capsulatum virulence is achieved, in part, through diverse and dynamic alterations to the fungal cell surface. Surface components associated with H. capsulatum pathogenicity include carbohydrates, lipids, proteins, and melanins. Here, we describe the various structures comprising the cell surface of H. capsulatum that have been associated with virulence and discuss their involvement in the pathobiology of disease.
PMCID: PMC3220077  PMID: 22121356
Histoplasma capsulatum; surface; cell wall; architecture; proteins; carbohydrates
24.  The inhibitory effect of cerulenin to yeasts is fungicidal 
Fatty acid biosynthesis plays a significant role in the growth and survival of diverse organisms. In yeasts, the de novo fatty acid synthesis (FAS) pathway produces and regulates essential fatty acid species such as saturated (SFA) and unsaturated (UFA) fatty acids that are required for generation and maintenance of cell membranes. Inhibition of enzymes in this pathway, such as fatty acid synthase and fatty acid desaturase, impede yeast cell growth unless appropriate exogenous fatty acids are provided.1,2 Although, the fatty acid biosynthesis pathway is essential to yeast cells, exploration of this pathway for combating fungal infections has been largely neglected. We and others have shown that deletion of a fatty acid synthase dramatically attenuates the virulence of the yeast Candida parapsilosis 2 and Candida albicans.1 Significantly, our data has revealed that inhibition of FAS enzymes results in the hypersensitivity of the yeast to serum, indicating that targeting this pathway is potentially an ideal way to combat systemic yeast infections.2 We demonstrated that using the minimal inhibitory concentration of cerulenin, a fatty acid synthase inhibitor, we could kill the wild type yeast cells in serum.2 Thus, the inhibitory effect of cerulenin (ie. blockade of the FAS pathway) on the yeast cells is fungicidal.
PMCID: PMC3306321  PMID: 22448300
25.  The Stearoyl-Coenzyme A Desaturase 1 Is Essential for Virulence and Membrane Stress in Candida parapsilosis through Unsaturated Fatty Acid Production ▿  
Infection and Immunity  2010;79(1):136-145.
Unsaturated fatty acids (UFA) are essential components of cells. In Saccharomyces cerevisiae, stearoyl-coenzyme A (CoA) desaturase 1 (OLE1) affects cell viability through the regulation of oleic (18:1) or palmitoleic (16:1) acid production. In this study, we used a targeted gene deletion approach to determine the impact of OLE1 on the emerging human pathogenic fungus Candida parapsilosis. We found that the deletion of OLE1 resulted in an auxotrophic yeast strain (designated OLE1 KO) that required unsaturated fatty acids for growth but not saturated fatty acids. Additionally, the production of UFA by OLE1 KO yeast cells was markedly reduced, suggesting that Ole1 is essential for UFA production. In contrast to wild-type C. parapsilosis, which produced pseudohyphal growth on UFA-supplemented medium agar, pseudohyphal formation in the OLE1 KO cells was severely impaired, suggesting that Ole1 regulates morphology. Furthermore, the OLE1 KO cells were hypersensitive to various stress-inducing factors, such as salts, SDS, and H2O2, especially at the physiological temperature. The results indicate that OLE1 is essential for the stress response, perhaps through the production of UFA for cell membrane biosynthesis. The OLE1 KO cells also were hypersensitive to human and fetal bovine serum, suggesting that targeting Ole1 could suppress the dissemination of yeast cells in the bloodstream. Murine-like macrophage J774.16 more efficiently killed the OLE1 KO yeasts, and significantly larger amounts of nitric oxide were detected in cocultures of macrophages and OLE1 KO cells than with wild-type or heterozygous strains. Moreover, the disruption of OLE1 significantly reduced fungal virulence in systemic murine infection. Taken together, these results demonstrate that Ole1 regulates the pathobiology of C. parapsilosis via UFA and that the OLE1 pathway is a promising antifungal target.
PMCID: PMC3019882  PMID: 20974817

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