An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal target from Candida albicans.

BACKGROUND: Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking. RESULTS: We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and ß-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports. CONCLUSIONS: The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.

Role of Protein Mannosylation in the Candida tropicalis-Host Interaction.

Mannans are components of the fungal wall attached to proteins via N- or O-linkages. In Candida albicans, Och1 is an α1,6-mannosyltransferase that adds the first mannose unit to the N-linked mannan outer chain; whereas Pmr1 is an ion pump that imports Mn2+ into the Golgi lumen. This cation is the cofactor of Golgi-resident mannosyltransferases, and thus Pmr1 is involved in the synthesis of both N- and O-linked mannans. Since we currently have limited information about the genetic network behind the Candida tropicalis protein mannosylation machinery, we disrupted OCH1 and PMR1 in this organism. The C. tropicalis pmr1Δ and och1Δ mutants showed increased doubling times, aberrant colony and cellular morphology, reduction in the wall mannan content, and increased susceptibility to wall perturbing agents. These changes were accompanied by increased exposure of both ß1,3-glucan and chitin at the wall surface of both mutant strains. Our results showed that O-linked mannans are dispensable for cytokine production by human mononuclear cells, but N-linked mannans and ß1,3-glucan are key ligands to trigger cytokine production in a co-stimulatory pathway involving dectin-1 and mannose receptor. Moreover, we found that the N-linked mannan core found on the surface of C. tropicalis och1Δ null mutant was capable of inducing cytokine production; and that a mannan-independent pathway for IL-10 production is present in the C. tropicalis-mononuclear cell interaction. Both mutant strains showed virulence attenuation in the Galleria mellonella and the mouse model of systemic candidiasis. Therefore, mannans are relevant for cell wall composition and organization, and for the C. tropicalis-host interaction.

Relative efficiency of Gafchromic EBT3 and MD-V3 films exposed to low-energy photons and its influence on the energy dependence.

Energy-dependence of Gafchromic films exposed to low-energy photons has been reported to be a function of absorbed-dose. However, these studies are based on a relative-response, R, which considers the absorbed-dose in water and not within the film sensitive-volume. This work investigated the relative-efficiency, REfilm, (ratio of absorbed-dose required to produce the same net optical density (netOD) by 60Co gamma and by x-ray) of Gafchromic EBT3 and MD-V3 films exposed to five x-ray beams from 20 kV to 160 kV and 60Co gamma rays. A factor that accounts for the energy-dependence, fx,Q,med, based on REfilm, phantom-material and depth at which the films are placed during irradiation was used to remove the influence of absorbed dose. Values of REfilm indicated that the absorbed dose from 60Co gamma rays needs to be 4 and 3 times larger than those from 20 kV x-rays to produce the same netOD within the EBT3 and MD-V3 sensitive volumes, respectively. Thus, saturation could help explain why Gafchromic films show under-response to very low doses from low-energy photon beams, regardless of film model. Furthermore, REfilm, was found to be nearly independent of netOD and colour-channels. Consequently, fx,Q,med is independent of the absorbed dose and colour-channels. In contrast, besides the variation with the photon energy, fx,Q,med varied with film model, depth and phantom material used during the irradiation. Thus, the results suggest that fx,Q,med is a more reliable wide-ranging parameter for evaluating the degree of energy-dependence of the film rather than the relative-response method commonly considered.

Role of protein phosphomannosylation in the Candida tropicalis-macrophage interaction.

Candida tropicalis is an opportunistic fungal pathogen responsible for mucosal and systemic infections. The cell wall is the initial contact point between a fungal cell and the host immune system, and mannoproteins are important components that play key roles when interacting with host cells. In Candida albicans, mannans are modified by mannosyl-phosphate moieties, named phosphomannans, which can work as molecular scaffolds to synthesize ß1,2-mannooligosaccharides, and MNN4 is a positive regulator of the phosphomannosylation pathway. Here, we showed that C. tropicalis also displays phosphomannans on the cell surface, but the amount of this cell wall component varies depending on the fungal strain. We also identified a functional ortholog of CaMNN4 in C. tropicalis. Disruption of this gene caused depletion of phosphomannan content. The C. tropicalis mnn4Δ did not show defects in the ability to stimulate cytokine production by human mononuclear cells but displayed virulence attenuation in an insect model of candidiasis. When the mnn4Δ-macrophage interaction was analyzed, results showed that presence of cell wall phosphomannan was critical for C. tropicalis phagocytosis. Finally, our results strongly suggest a differential role for phosphomannans during phagocytosis of C. albicans and C. tropicalis.

Functional characterization of the Sporothrix schenckii Ktr4 and Ktr5, mannosyltransferases involved in the N-linked glycosylation pathway.

Sporothrix schenckii is one of the causative agents of the deep-seated mycosis sporotrichosis, a fungal infection with worldwide distribution. Fungus-specific molecules and biosynthetic pathways are potential targets for the development of new antifungal drugs. The MNT1/KRE2 gene family is a group of genes that encode fungus-specific Golgi-resident mannosyltransferases that participate in the synthesis of O-linked and N-linked glycans. While this family is composed of five and nine members in Candida albicans and Saccharomyces cerevisiae, respectively, the S. schenckii genome contains only three putative members. MNT1 has been previously characterized as an enzyme that participates in the synthesis of both N-linked and O-linked glycans. Here, we aimed to establish the functional role of the two remaining family members, KTR4 and KTR5, in the protein glycosylation pathways by using heterologous complementation in C. albicans mutants lacking genes of the MNT1/KRE2 family. The two S. schenckii genes restored defects in the elaboration of N-linked glycans, but no complementation of mutants that synthesize truncated O-linked glycans was observed. Therefore, our results suggest that MNT1 is the sole member with a role in O-linked glycan elaboration, whereas the three family members have redundant activity in the S. schenckii N-linked glycan synthesis.

The Sporothrix schenckii Gene Encoding for the Ribosomal Protein L6 Has Constitutive and Stable Expression and Works as an Endogenous Control in Gene Expression Analysis.

Sporothrix schenckii 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, ß-actin, ß-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 S. schenckii infected larvae of Galleria mellonella 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 N-linked glycosylation pathway, a histidine kinase whose expression is regulated during the fungal dimorphism, and a glycosidase that participates in sucrose assimilation.

Saccharomyces cerevisiae KTR4, KTR5 and KTR7 encode mannosyltransferases differentially involved in the N- and O-linked glycosylation pathways.

Saccharomyces cerevisiae is a model to understand basic aspects of protein glycosylation pathways. Although these metabolic routes have been thoroughly studied, there are still knowledge gaps; among them, the role of the MNT1/KRE2 gene family. This family is composed of nine members, with only six functionally characterized. The enzymes Ktr1, Ktr3, and Mnt1/Kre2 have overlapping activities in both O-linked and N-linked glycan synthesis; while Ktr2 and Yur1 participate exclusively in the elongation of the N-linked glycan outer chain. KTR6 encodes for a phosphomannosyltransferase that synthesizes the cell wall phosphomannan. Here, we aimed to establish the functional role of KTR4, KTR5 and KTR7 in the protein glycosylation pathways, by using heterologous complementation in Candida albicans null mutants lacking members of the MNT1/KRE2 gene family. The three S. cerevisiae genes restored defects in the C. albicans N-linked glycosylation pathway. KTR5 and KTR7 partially complemented a C. albicans null mutant with defects in the synthesis of O-linked glycans, and only KTR4 fully elongated the O-linked glycans like wild-type cells. Therefore, our results suggest that the three genes have a redundant activity in the S. cerevisiae N-linked glycosylation pathway, but KTR4 plays a major role in O-linked glycan synthesis.

Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence.

The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of ß1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and ß1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and ß1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O-linked mannans.

Coordination and speciation of cadmium in corn seedlings and its effects on macro- and micronutrients uptake.

The effect of cadmium (Cd) on both the absorption of important nutrients and the synthesis of low molecular weight thiols (LMWTs) was investigated in corn plants. The inductively coupled plasma-optical emission spectroscopy results demonstrated that the concentration of Cd in tissues (mainly in roots) increased as the concentration in the medium increased. In addition, the concentration of phosphorus increased in roots of Cd treated plants but remained at normal concentration in shoots. On the other hand, the uptake of sulfur (S) followed a similar trend as the Cd uptake. The concentration of S and the production of LMWT were found to increase significantly upon exposure to Cd. The results of the X-ray absorption spectroscopy analyses indicated that Cd within tissues was bound to S ligands with interatomic distances of 2.51-2.52 A. These results confirm a strong linkage between S uptake and the production of LMWT upon exposure to Cd.