Mannans are components of the fungal wall attached to proteins via
- or
-linkages. In
, Och1 is an α1,6-mannosyltransferase that adds the first mannose unit to the
-linked mannan outer chain; whereas ...Pmr1 is an ion pump that imports Mn
into the Golgi lumen. This cation is the cofactor of Golgi-resident mannosyltransferases, and thus Pmr1 is involved in the synthesis of both
- and
-linked mannans. Since we currently have limited information about the genetic network behind the
protein mannosylation machinery, we disrupted
and
in this organism. The
Δ and
Δ 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 β1,3-glucan and chitin at the wall surface of both mutant strains. Our results showed that
-linked mannans are dispensable for cytokine production by human mononuclear cells, but
-linked mannans and β1,3-glucan are key ligands to trigger cytokine production in a co-stimulatory pathway involving dectin-1 and mannose receptor. Moreover, we found that the
-linked mannan core found on the surface of
Δ null mutant was capable of inducing cytokine production; and that a mannan-independent pathway for IL-10 production is present in the
-mononuclear cell interaction. Both mutant strains showed virulence attenuation in the
and the mouse model of systemic candidiasis. Therefore, mannans are relevant for cell wall composition and organization, and for the
-host interaction.
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.
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.
The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast
, 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
homolog in a low-virulent species that rarely causes candidiasis,
. We disrupted
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 β1,3-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 β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate
sensing by monocytes was critically dependent on the recognition of
-linked mannans and β1,3-glucan, as reported in other
species. In addition, chemical remotion of cell wall
-linked mannans was found to positively influence the recognition of
by human monocytes, suggesting that
-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in
. Finally, mice infected with
Δ 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
infection model. This study thus demonstrates that mannans are relevant for the
-host interaction, with an atypical role for
-linked mannans.
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 β1,2-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.
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.
Abstract
We present the first results from the ongoing, intensive, multiwavelength monitoring program of the luminous Seyfert 1 galaxy Mrk 817. While this active galactic nucleus was, in part, ...selected for its historically unobscured nature, we discovered that the X-ray spectrum is highly absorbed, and there are new blueshifted, broad, and narrow UV absorption lines, which suggest that a dust-free, ionized obscurer located at the inner broad-line region partially covers the central source. Despite the obscuration, we measure UV and optical continuum reverberation lags consistent with a centrally illuminated Shakura–Sunyaev thin accretion disk, and measure reverberation lags associated with the optical broad-line region, as expected. However, in the first 55 days of the campaign, when the obscuration was becoming most extreme, we observe a de-coupling of the UV continuum and the UV broad emission-line variability. The correlation recovered in the next 42 days of the campaign, as Mrk 817 entered a less obscured state. The short C
iv
and Ly
α
lags suggest that the accretion disk extends beyond the UV broad-line region.
Although the WHO strategy aims to eliminate the hepatitis C virus (HCV) as a public health threat by 2030, national strategies are variable worldwide. This study aimed to assess the establishment of ...different policies and strategies to eliminate HCV in the Americas.
We conducted a 23-item survey about HCV-related policies and strategies among gastroenterologists and hepatologists in the Americas. The survey was carried out electronically (2022–2023). Data were compared with governmental institutions, regulatory agencies, scientific societies, and scientific publications. We estimated an index obtained from a regression scoring method through exploratory analysis, and row values were normalized from 0 to 100.
We obtained 52 responses from 19 countries. The median HCV-related policies index was 51.4 IQR:27.3–70.1. The lower establishment of HCV-related policies was observed in Ecuador (0.0), Honduras (6.6), and Costa Rica (9.8), while the highest performance was observed in Argentina (94.1), Colombia (94.7), and Canada (100)(Figure 1A). Fifteen (78.9%) countries have adopted a national strategic plan to eliminate HCV. Three (15.8%) countries have universal screening for HCV infection (Figure 1B). After a positive HCV serological test, 10 (52.6%) countries perform reflex testing to confirm HCV diagnosis using the same sample. However, only 7 (36.8%) countries have an alert system for the requesting physician. Twelve (63.2%) countries have a direct referral system for specialized care of HCV-positive cases. Universal access to direct-acting antivirals (DAAs) exists in 15 (78.9%) countries. Universal access to DAAs was not widely available in Cuba, Ecuador, Venezuela, and the United States. Seven (36.8%) countries have generic DAAs available. Only 3 (15.8%) countries performed a retrospective search for HCV-positive cases that could have been lost to follow-up.
Although most countries have adopted a national strategic plan to eliminate HCV, there are several issues and barriers to elimination in the Americas.
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