Diana Bridon da Graça Sgarbi

Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii

Ergosterol peroxide, a presumed product of the H_2O_2-dependent enzymatic oxidation of ergosterol, has been isolated from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (1H and 13C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C_28H_44O_3, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract.

DETECTION OF CELLULAR-IMMUNITY WITH THE SOLUBLE-ANTIGEN OF THE FUNGUS SPOROTHRIX-SCHENCKII IN THE SYSTEMIC FORM OF THE DISEASE

Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions. The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses. In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S. schenckii. The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks. The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals). Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen. This depression frequently indicates worsening of the disease, with greater involvement of the host. This is a promising field of research for a better understanding of the pathogeny of this mycosis.

Current Research on the Immune Response to Experimental Sporotrichosis

Sporotrichosis is often manifested as a chronic granulomatous infection and the monocytes/macrophages play a central role in the host defense system. Surface components of Sporothrix schenckii have been characterized and suggestions have been made as to their possible role in pathogenicity. Ergosterol peroxide, cell-wall compounds (alkali-insoluble fraction-F1 and lipid extract-LEY), and exoantigen from the yeast form of the fungus have been characterized as virulence factors, activating both innate, by cytotoxins linked to the activation of reactive oxygen and nitrogen species (H2O2 and NO), and adaptive immune response to produce cytokines Th1 and Th2 profile. In this study, preliminary results have demonstrated that, in systemic sporotrichosis, TLR-4 triggers the innate immune response, activating an oxidative burst. These data represent the first report of the participation of TLR-4 in murine sporotrichosis, in the presence of lipids from the cell wall of S. schenckii. These results taken together may open new perspectives of study leading to an antifungal agent that could be used to benefit the entire population.

Response of macrophage Toll-like receptor 4 to a Sporothrix schenckii lipid extract during experimental sporotrichosis

Toll‐like receptors have been implicated in the recognition of various pathogens, including bacteria, viruses, protozoa and fungi. However, no information is available about Toll‐like receptor 4 (TLR4) participation in Sporothrix schenckii recognition and the consequent triggering of the immune response to this fungal pathogen. Following activation of TLRs by ligands of microbial origin, several responses are provoked, including reactions in immune cells that may lead them to produce signalling factors that trigger inflammation. The present study was designed to elucidate the role of TLR4 during the host response to S. schenckii. TLR4‐deficient (C3H/HeJ) and control mice (C3H/HePas) were infected with S. schenckii yeast cells and immune response was assessed over 10 weeks by assaying production of pro‐inflammatory mediator (nitric oxide and tumour necrosis factor‐α) and anti‐inflammatory cytokine (interleukin‐10) by peritoneal macrophages and their correlation with apoptosis in peritoneal exudate cells. We found that both pro‐inflammatory and anti‐inflammatory mediators are reduced in TLR4‐deficient mice, suggesting the involvement of this receptor in the recognition of this infectious agent. Translocation into the nucleus of nuclear transcription factor, nuclear factor‐κB, was also evaluated and showed higher levels in TLR‐4 normal mice, consistent with the results found for cytokine production. We are showing here, for the first time, the involvement of TLR4 in S. schenckii recognition. Taken together, our results demonstrate that the activation of peritoneal macrophages in response to S. schenckii lipid extracts has different responses in these two mouse strains which differ in TLR4 expression, suggesting an important role for TLR4 in governing the functions of macrophages in this fungal infection.

Host organism defense by a peptide-polysaccharide extracted from the fungus Sporothrix schenckii

A peptide-polysaccharide, a peptide-rhamnomannan, was isolated from the pathogenic yeast form of the fungus Sporothrix schenckii. This substance, which may play a role in fungal virulence, was tested in an animal model of systemic disease, and depression of the immune response was observed in the animals between the 4th and 6th week of infection. Concomitantly, this compound showed mitogenic activity when challenged with normal lymphocytes and was also found to be involved in the inflammatory response. These results provide further information for the understanding of fungal implantation in tissues and of the pathogenicity of this systemic mycosis.

Candida haemulonii complex: species identification and antifungal susceptibility profiles of clinical isolates from Brazil

OBJECTIVES The emerging fungal pathogens comprising the Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii) are notable for their antifungal resistance. Twelve isolates with phenotypic similarity to C. haemulonii were recovered from patients in Brazilian hospitals. Here we aimed to identify these isolates by a molecular approach, using the current classification of this fungal complex, and to evaluate their antifungal susceptibility profiles. METHODS The fungal isolates were rechecked to certify their authentication by mycology methodologies and then characterized by ITS1-5.8S-ITS2 gene sequencing. A susceptibility assay was performed using the broth microdilution method published by CLSI (M27-A3/M27-S3). RESULTS Based on biochemical tests, all Brazilian isolates were identified as C. haemulonii. After employing ITS sequencing, five isolates were identified as C. haemulonii, four as C. duobushaemulonii and three as C. haemulonii var. vulnera. All 12 clinical isolates were resistant to amphotericin B (MICs ranged from 2 to >16 mg/L) and fluconazole (MICs ≥ 64 mg/L). One isolate of C. haemulonii var. vulnera and two isolates of C. duobushaemulonii were susceptible-dose dependent to itraconazole, while the remaining isolates (75%) were resistant to this antifungal. Eight out of 12 isolates (66.7%) were resistant to voriconazole (MICs ≥ 16 mg/L), while all isolates were susceptible to caspofungin (MICs ≤ 0.5 mg/L). CONCLUSIONS Our results reinforce the importance of molecular identification in differentiating species of the C. haemulonii complex. Moreover, the antifungal multiresistant profile of clinical isolates of the C. haemulonii complex represents a challenge to the treatment of such infections.

Protease and phospholipase activities of Candida spp. isolated from cutaneous candidiasis

BACKGROUND Cases of superficial and invasive mycoses caused by emerging species of Candida have been increasingly reported over the last thirty years. The production of hydrolytic enzymes plays a central role in the fungal infective process. In Candida infections the secretion of both proteases and phospholipases are well-known virulence attributes. AIMS To determine the protease and phospholipase production from 58 human clinical isolates of Candida obtained from individuals with cutaneous candidiasis seen in the Human and Veterinary Diagnostic Mycology Sector from Universidade Federal Fluminense (UFF), Brazil, from November 2008 to August 2009. METHODS Fungal identification was performed using biochemical tests. Proteolytic activity was detected on agar plates containing bovine serum albumin, and phospholipase production was determined on egg-yolk plates. RESULTS The Candida species isolated were Candida parapsilosis (27.59%), Candida famata (18.96%), Candida albicans (15.52%), Candida haemulonii (12.06%), Candida ciferri (8.62%), Candida guilliermondii (6.90%), Candida tropicalis (5.17%) and Candida lipolytica (5.17%). All isolates of C. albicans produced both protease and phospholipase. As regards the isolates of non-C. albicans Candida species, 53.06% and 4.08% were able to produce protease and phospholipase, respectively. For example, the majority of isolates of C. parapsilosis (15/16) produced protease, while 40% of C. ciferri isolates (2/5) were phospholipase producers. This study shows, for the first time, that C. ciferri and C. haemulonii strains were able to produce protease. CONCLUSIONS Collectively, our results showed that different species of Candida isolated from cutaneous lesions were able to produce proteases and/or phospholipases, which are multifunctional molecules directly involved in the infectious process of these fungi.

Environmental Conditions and Fungal Pathogenicity

The different species of the so-called Sporothrix complex are environmental fungus found in soils, plants, water, decaying vegetables, and other outdoor environments. Although they have been isolated from diverse locations, including contaminated areas, few studies have addressed the influence of the environment on the virulence of these pathogens. However, some researches in S. schenckii and other similar pathogenic fungi, suggest that adverse conditions in the natural habitat can trigger the expression of different virulence factors that confer survival advantages both in the environment and in host tissues. In this chapter, we approach advances in understanding of the biology of S. schenckii and how environmental factors can modify its virulence.