Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has ...severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology.
Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known ...that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.
Abstract
The description of cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. Sporothrix schenckii causes a benign chronic subcutaneous mycosis, ...Sporothrix brasiliensis is highly virulent, and Sporothrix globosa mainly causes fixed cutaneous lesions. Furthermore, S. brasiliensis is the prevalent species related to cat-transmitted sporotrichosis. Sources of infection, transmission, and distribution patterns also differ between species, and variability differs between species because of different degrees of clonality. The present review article will cover several aspects of the biology of clinically relevant agents of sporotrichosis, including epidemiological aspects of emerging species. Genomic information of Sporothrix spp. is also discussed. The cell wall is an essential structure for cell viability, interaction with the environment, and the host immune cells and contains several macromolecules involved in virulence. Due to its importance, aspects of glycosylation and cell wall polysaccharides are reviewed. Recent genome data and bioinformatics analyses helped to identify specific enzymes of the biosynthetic glycosylation routes, with no homologs in mammalian cells, which can be putative targets for development of antifungal drugs. A diversity of molecular techniques is available for the recognition of the clinically relevant species of Sporothrix. Furthermore, antigens identified as diagnostic markers and putative vaccine candidates are described. Cell-mediated immunity plays a key role in controlling infection, but Sporothrix species differ in their interaction with the host. The adaptive branch of the immune response is essential for appropriate control of infection.
In this study, the human immune response mechanisms against
and
, two causative agents of human and animal sporotrichosis, were investigated. The interaction of
and
with human monocyte-derived ...macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of
yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two
yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the
yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both
and
, suggesting that the host complement system plays an essential role in the inflammatory immune response against these
species. Nevertheless, the structural differences in the PRMs of the two
species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and
CR3 receptor mediating an inflammatory response to
species.
and
are usually associated to sporotrichosis, a subcutaneous mycosis worldwide distributed. Comparative analyses between these two species indicate they contain genetic and physiological differences ...that are likely to impact the interaction with host cells. Here, we study the composition of the cell wall from conidia, yeast-like cells and germlings of both species and found they contained the same sugar composition. The carbohydrate proportion in the
wall was similar across the three cell morphologies, with exception in the chitin content, which was significantly different in the three morphologies. The cell wall from germlings showed lower rhamnose content and higher glucose levels than other cell morphologies. In
, the wall sugars were constant in the three morphologies, but glucose was lower in yeast-like cells. In
cells most of chitin and β1,3-glucan were underneath wall components, but in
germlings, chitin was exposed at the cell surface, and β1,3-glucan was found in the outer part of the conidia wall. We also compared the ability of these cells to stimulate cytokine production by human peripheral blood mononuclear cells. The three
morphologies stimulated increased levels of pro-inflammatory cytokines, when compared to
cells; while the latter, with exception of conidia, stimulated higher IL-10 levels. Dectin-1 was a key receptor for cytokine production during stimulation with the three morphologies of
, but dispensable for cytokine production stimulated by
germlings. TLR2 and TLR4 were also involved in the sensing of
cells, with a major role for the former during cytokine stimulation. Mannose receptor had a minor contribution during cytokine stimulation by
yeast-like cells and germlings, but
conidia and
yeast-like cells stimulated pro-inflammatory cytokines via this receptor. In conclusion,
and
, have similar wall composition, which undergoes changes depending on the cell morphology. These differences in the cell wall composition, are likely to influence the contribution of immune receptors during cytokine stimulation by human monocytes.
We herein present a Brazilian guideline for the management of feline sporotrichosis, a mycosis caused by
Sporothrix brasiliensis.
This guideline is an effort of a national technical group organized ...by the Working Group on
Sporothrix
and Sporotrichosis of the International Society for Human and Animal Mycology (ISHAM). This publication intends to provide information on clinical-epidemiological aspects of this zoonosis, as well as a literature revision. Moreover, it gives some practical information on diagnosis and treatment of feline sporotrichosis. It also contains information that can be helpful for the prevention and control of
S. brasiliensis
transmission
.
Sporothrix schenckii
,
Sporothrix brasiliensis
, and
Sporothrix globosa
are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated ...with each species but remain largely uncharacterized. The
S. schenckii
and
S. brasiliensis
cell wall composition and virulence are influenced by the culturing media, with little or no influence on
S. globosa
. By keeping constant the culturing media, we compared the cell wall composition of three
S. schenckii
and two
S. brasiliensis
strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five
Sporothrix
spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-β-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-β-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in
Sporothrix
spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.
Monoclonal antibodies (mAbs) are promising alternatives to treat infectious diseases, especially given their potential for applications in combination therapies with antimicrobial drugs to enhance ...the antifungal efficacy. Protection mediated by mAbs used to treat experimental paracoccidioidomycosis (PCM) has been demonstrated previously. Our aim in the present work was to characterize a monoclonal antibody (mAbF1.4) raised against a cell wall glycoconjugate fraction of
spp. and to analyze its efficacy combined with trimethoprim-sulfamethoxazole (TMP/SMX) as treatment for experimental PCM. We demonstrated that the epitope recognized by mAbF1.4 is consistent with branched glucose residues present on a cell wall β-glucan polymer.
, mAbF1.4 increased the phagocytic capacity and nitric oxide concentration induced by the macrophage cell line J774.1A, and this resulted in a significant reduction in the viability of the opsonophagocytized yeasts.
, we detected a significant reduction in pulmonary fungal burdens of mice treated with mAbF1.4 in association with TMP/SMX, which correlated with increased pulmonary concentrations (determined by ELISA) of IFN- γ, TNF-α, IL-10 and IL-17. In parallel, we observed a decrease in IL-4, suggesting that the treatment was associated with a mixed Th1-Th17 type immune response. Histopathology of lung segments from mice receiving the combination therapy showed a significant reduction in granulomas, which were well-defined, and improved maintenance of lung architecture. These findings demonstrate that mAbF1.4 + TMP/SMX therapy is a promising approach to combat PCM as well as decrease disease sequelae and highlights the potential benefits of immune mediators in PCM combined immunotherapy.
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•This is the first study on the purification of Cassia leiandra trypsin inhibitor (ClTI).•ClTI is a Kunitz-type inhibitor composed of a single 19,484Da polypeptide chain.•ClTI is an ...uncompetitive inhibitor with a Ki of 6.25×10−8M.•ClTI inhibits Aedes aegypti larval midgut proteases.•ClTI impairs the survival and larval cycle of Aedes aegypti (LC50 of 2.28×10−2M).
A trypsin inhibitor from Cassia leiandra seeds, named ClTI, was purified, characterized, and its insecticidal activity against Aedes aegypti evaluated. ClTI was purified by DEAE-Cellulose and trypsin-Sepharose 4B chromatography, with a 15.5-fold purification and 2.4% yield. ClTI is composed of a 19,484Da polypeptide chain as revealed by mass spectrometry, it is not a glycoprotein, its amino acid sequence is similar to other Kunitz-type inhibitors, and it comprises 35% β-sheets, 14% β-turns, and 50% disordered secondary structures. ClTI is an uncompetitive inhibitor of bovine trypsin (IC50 of 33.81×10−8M, Ki of 6.25×10−8M) stable over a broad range of pHs (2.2–10.0) and temperatures (30–70°C), but dithiothreitol led to a partial loss of the inhibitory activity. ClTI, at 4.65×10−6M, reduced in 50% the activity of the Ae. aegypti midgut proteases. ClTI also promoted acute toxicity on the 3rd instar larvae of Ae. aegypti, with an LC50 of 2.28×10−2M. Moreover, it caused a 24-h delay of the larvae development and 44% mortality after ten days of exposure. Altogether, these results suggest that ClTI has potential as a natural compound to control Ae. aegypti, a vector of several infection diseases.
Sporotrichosis is a neglected zoonosis caused by pathogenic fungi belonging to the Sporothrix schenckii complex. In Rio de Janeiro state, this disease reached an epidemic status with over 4700 ...domestic felines and around 4000 humans affected since the mid-90s. The present study evaluated clinical and epidemiological aspects and also the frequency of colonization and infection by these fungi in healthy cats and among those with suspicious cutaneous lesions, inhabiting four Rio de Janeiro state distinct areas.
Three hundred and seventy-one cats were included in two groups: 175 healthy cats CRG and 196 cats showing lesions suggesting sporotrichosis SSG. Mycological diagnosis allowed SSG animals to be divided in positive 104 cats; +SG and negative 92 cats; -SG groups. Nails, oral mucosa and lesions swabs were submitted to culture and potential colonies were subculture for micromorphologycal analysis, dimorphism and molecular tests. In the CRG, only one cat was colonized in the oral cavity 0.57%; in the -SG group, four animals showed colonization of the nail and/or oral cavity 4.3%; while the highest frequency of colonization 39.4% was observed in the +SG. All molecularly typed isolates were identified as S. brasiliensis.
The results obtained here indicate that healthy cats have a minor role in sporotrichosis transmission within the state of Rio de Janeiro. Conversely, a higher participation of diseased feline in sporotrichosis transmission was evidenced, especially by the colonization of their oral cavity. Sporothrix brasiliensis equally affects and colonizes animals from distinct Rio de Janeiro state areas. Thus, we hypothesize that sporotrichosis is a uniform endemic throughout the state, whose transmission depends mainly on the contact with cats with sporotrichosis. Since Rio de Janeiro displays a world unique epidemic model of the disease, not fully understood, data on the infected and non-infected animals can be of major importance for future strategies of sporotrichosis prevention and control. Finally, considering the importance of the current concept of "one health", the experience here observed can be helpful for distinct epizootias and/or zoonosis.