Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. ...However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns.
is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The
immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the
immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with
,
,
,
, and
. The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.
is one of the causative agents of sporotrichosis, a worldwide-distributed mycosis that affects humans and other mammals. The interest in basic and clinical features of this organism has significantly ...increased in the last years, yet little progress in molecular aspects has been reported. Gene expression analysis is a set of powerful tools that helps to assess the cell response to changes in the extracellular environment, the genetic networks controlling metabolic pathways, and the adaptation to different growth conditions. Most of the quantitative methodologies used nowadays require data normalization, and this is achieved measuring the expression of endogenous control genes. Reference genes, whose expression is assumed to suffer minimal changes regardless the cell morphology, the stage of the cell cycle or the presence of harsh extracellular conditions are commonly used as controls in Northern blotting assays, microarrays, and semi-quantitative or quantitative RT-PCR. Since the biology of the organisms is usually species specific, it is difficult to find a reliable group of universal genes that can be used as controls for data normalization in experiments addressing the gene expression, regardless the taxonomic classification of the organism under study. Here, we compared the transcriptional stability of the genes encoding for elongation factor 1A, Tfc1, a protein involved in transcription initiation on Pol III promoters, ribosomal protein L6, histone H2A, β-actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase, UAF30, the upstream activating factor 30, and the transcription initiation factor TFIID subunit 10, during the fungal growth in different culture media and cell morphologies. Our results indicated that only the gene encoding for the ribosomal protein L6 showed a stable and constant expression. Furthermore, it displayed not transcriptional changes when
infected larvae of
or interacted with immune cells. Therefore, this gene could be used as control for data normalization in expression assays. As a proof of concept, this gene was used to assess the expression of genes encoding for glycosidases involved in the protein
-linked glycosylation pathway, a histidine kinase whose expression is regulated during the fungal dimorphism, and a glycosidase that participates in sucrose assimilation.
Human fungal infections remain a major challenge in medicine. Only a limited number of antifungal drugs are available, which are often related to severe adverse effects. In addition, there is an ...increased emergence related to resistant strains, which makes imperative to understand the host-pathogen interactions as well as to develop alternative treatments. Host innate and adaptive immunity play a crucial role controlling fungal infections; therefore, vaccines are a viable tool to prevent and treat fungal pathogens. Innate immunity is triggered by the interaction between the cell surface pattern recognition receptors (PRRs) and the pathogen-associated molecular patterns (PAMPs). Such an initial immunological response is yet little understood in fungal infections, in part due to the complexity and plasticity of the fungal cell walls. Described host cell-fungus interactions and antigenic molecules are addressed in this paper. Furthermore, antigens found in the cell wall and capsule, including peptides, glycoproteins, glycolipids, and glycans, have been used to trigger specific immune responses, and an increased production of antibodies has been observed when attached to immunogenic molecules. The recent biotechnological advances have allowed the development of vaccines against viral and bacterial pathogens with positive results; therefore, this technology has been applied to develop anti-fungal vaccines. Passive immunization has also emerged as an appealing alternative to treat disseminated mycosis, especially in immunocompromised patients. Those approaches have a long way to be seen in clinical cases. However, all studies discussed here open the possibility to have access to new therapies to be applied alone or in combination with current antifungal drugs. Herein, the state of the art of fungal vaccine developments is discussed in this review, highlighting new advances against
,
, and
spp.
Sporotrichosis is an infection caused by members of the Sporothrix genus, and among them, Sporothrix schenckii is one of the etiological agents. Both, the disease and the causative agent have gained ...interest in the recent years, because of the report of epidemic outbreaks, and the description of the disease transmission from animals to human beings. Despite the relevance of S. schenckii in the clinical field, there are basic aspects of its biology poorly explored. So far, Agrobacterium tumefaciens-mediated transformation has been reported as an alternative for genetic manipulation of this fungal pathogen. Here, we report the optimization of the transformation method and used this to generate insertional mutants that express the green fluorescent protein in S. schenckii. We obtained five mutant strains that showed mitotic stability and expression of the reporter gene. The strains displayed normal cell wall composition, and a similar ability to interact ex vivo with human monocytes and monocyte-derived macrophages. Moreover, the virulence in larvae of Galleria mellonella was similar to that obtained with the wild-type control strains. These data indicate that these fluorescent mutants with normal ability to interact with the host could be used in bioimaging to track the host-Sporothrix interaction in vivo.
•Agrobacterium-mediated transformation of Sopothrix schenckii was optimized.•Insertional mutants expressing GFP were generated.•The mutant strains showed normal cell wall composition and interaction with the host.•The GFP-expressing mutants are suitable for the study of the Sporothrix-host interaction.
is a neglected fungal pathogen for the human being and other mammals. In several fungal systems, Och1 is a Golgi α1,6-mannosyltransferase with a key function in the synthesis of
-linked glycans; ...which are important elements during the host-fungus interplay. The role of
in fungal virulence seems to be species-specific, being an essential component for
virulence and dispensable during the interaction of
with the host.
Here, we silenced
and characterized the phenotype of the mutant strains.
The mutant strains did not show defects in the cell or colony morphology, the growth rate or the ability to undergo dimorphism; but the cell wall changed in both composition and exposure of inner components at the surface. When interacting with human monocytes, the silenced strains had a reduced ability to stimulate TNFα and IL-6 but stimulated higher levels of IL-10. The interaction with human macrophages was also altered, with reduced numbers of silenced cells phagocytosed. These strains showed virulence attenuation in both
and in the mouse model of sporotrichosis. Nonetheless, the cytokine levels in infected organs did not vary significantly when compared with the wild-type strain.
Our data demonstrate that
silencing affects different aspects of the
-host interaction.
A series of complex boronic acids were prepared through multicomponent reactions (MCRs). Both Passerini and Ugi MCRs were carried out in which one component was an arylboronic acid. The resulting ...highly functionalized boronic acids participated efficiently in the Liebeskind–Srogl cross-coupling reaction with meso-methylthioBODIPY derivatives to yield complex borondipyrromethene (BODIPY) dyes in good yields. The joined spectroscopic and computational study points out the deep impact of the arylated chromophoric position on the photophysical signatures. Thus, unconstrained aryls grafted at the meso position did not sway the spectral band positions but switched on new nonradiative relaxation channels, whereas additional arylation at the opposite α-pyrrolic position softened such fluorescence quenching and shifted the emission to the red-edge of the visible spectrum. The conducted biological analysis revealed that peripheral blood mononuclear cells incubated with these new compounds showed reduced cytotoxicity and retained their normal activities. Additionally, the dyes remained stable inside the cells after 24 h of incubation. These results demonstrated that these novel fluorescent probes based on BODIPY can be applied for cell imaging and analysis, expanding their applications.
Candida albicans is the main causative agent of systemic candidiasis, a condition with high mortality rates. The study of the interaction between C. albicans and immune system components has been ...thoroughly studied and nowadays there is a model for the anti-C. albicans immune response; however, little is known about the sensing of other pathogenic species of the Candida genus. Sporothrix schenckii is the causative agent of sporotrichosis, a subcutaneous mycosis, and thus far there is limited information about its interaction with the immune system. In this paper, we review the most recent information about the immune sensing of species from genus Candida and S. schenckii. Thoroughly searches in scientific journal databases were performed, looking for papers addressing either Candida- or Sporothrix-immune system interactions. There is a significant advance in the knowledge of non-C. albicans species of Candida and Sporothrix immune sensing; however, there are still relevant points to address, such as the specific contribution of pathogen-associated molecular patterns (PAMPs) for sensing by different immune cells and the immune receptors involved in such interactions. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).
The production of total phenolic content (TPC) and total flavonoid content (TFC), antioxidant (AA), and copper chelating (CACu
+
) activities of
Rhizophora mangle
from three coastal lagoons under ...different grades of anthropogenic pollution and pollutant presence were analyzed.
R. mangle
is a mangrove plant that inhabits tropical coastal lagoons and shows the ability to accumulate pollutants in their tissues and respond to environmental stressors
via
the production of secondary metabolites AA and CACu
+
activities. In total, 108 samples (June 2010 to August 2011) of leaves, barks, and the roots of
R. mangle
from Magdalena Bay (MBAY), Navachiste Lagoon (NLAG), and Terminos Lagoon (TLAG) (Mexico) were collected. Folin-Ciocalteu colorimetric, aluminum chloride colorimetric, ABTS∙
+
discoloration, and pyrocatechol-violet methods calculated TPC, TFC, AA, and, CACu
+
, respectively. Atomic absorption spectrometry, gas chromatography, and Walkley–Black methods were used to calculate the metal content, organochlorine pesticides (OCPs), and organic matter (OM) in sediments. One-way ANOVA and
post hoc
Tukey’s, Scheffé’s, and Bonferroni and Holm multiple comparisons were used to determine significance, and Pearson’s test was applied to determine the coefficient of correlations. Significant differences among the lagoons of TPC, TFC, AA, and Cu
+
CA activities of
Rhizophora mangle
are explained by their different grades and type of anthropogenic pollution. A significant increase in the TPC and TFC in bark implies a response to the metal and OCPs pollution in surrounding sediments. Significant AA and Cu
+
CA activities responded to combat environmental stress conditions caused by the OCP and trace metal concentrations in surrounding sediments. Strong and positive correlations among OCP and trace metal were found and related to the constant pesticide residues input. However, OCP concentrations in the surrounding sediments of
R. mangle
trees did not influence the phenolic production of AA or the Cu+ CA.
R. mangle
is affected by the type of anthropogenic pollutants and was reflected in the bio-compounds production, antioxidant, and chelating activities related to the concentration of the metals in sediments.
Protein glycosylation pathways are conserved metabolic processes in eukaryotic organisms and are required for cell fitness. In fungal pathogens, the N-linked glycosylation pathway is indispensable ...for proper cell wall composition and virulence. In Sporothrix schenckii sensu stricto, the causative agent of sporotrichosis, little is known about this glycosylation pathway. Here, using a genome-wide screening for putative members of the glycosyl hydrolase (CAZy - GH) families 47 and 63, which group enzymes involved in the processing step during N-linked glycan maturation, we found seven homologue genes belonging to family 47 and one to family 63. The eight genes were individually expressed in C. albicans null mutants lacking either MNS1 (for members of family 47) or CWH41 (for the member of family 63). Our results indicate that SsCWH41 is the functional ortholog of CaCWH41, whereas SsMNS1 is the functional ortholog of CaMNS1. The remaining genes of family 47 encode Golgi mannosidases and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like proteins (EDEMs). Since these GH families gather proteins used as target for drugs to control cell growth, identification of these genes could help in the design of antifungals that could be used to treat sporotrichosis and other fungal diseases. In addition, to our knowledge, we are the first to report that Golgi mannosidases and EDEMs are expressed and characterized in yeast cells.
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