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The ability to undergo polarised cell growth is fundamental to the development of almost all walled organisms. Fungi are characterised by yeasts and moulds, and both cellular forms have been studied extensively as tractable models of cell polarity. Chitin is a hallmark component of fungal cell walls. Chitin synthesis is essential for growth, viability and rescue from many conditions that impair cell-wall integrity. In the polymorphic human pathogen Candida albicans, chitin synthase 3 (Chs3) synthesises the majority of chitin in the cell wall and is localised at the tips of growing buds and hyphae, and at the septum. An analysis of the C. albicans phospho-proteome revealed that Chs3 can be phosphorylated at Ser139. Mutation of this site showed that both phosphorylation and dephosphorylation are required for the correct localisation and function of Chs3. The kinase Pkc1 was not required to target Chs3 to sites of polarised growth. This is the first report demonstrating an essential role for chitin synthase phosphorylation in the polarised biosynthesis of fungal cell walls and suggests a new mechanism for the regulation of this class of glycosyl-transferase enzyme. [ABSTRACT FROM AUTHOR]

A soluble α-mannosidase from Candida albicans CAI-4 was purified by conventional methods of protein isolation. Analytical electrophoresis of the purified preparation revealed two polypeptides of 52 and 27 kDa, the former being responsible for enzyme activity. The purified, 52 kDa enzyme trimmed Man9GlcNAc2, producing Man8GlcNAc2 isomer B and mannose, and was inhibited preferentially by 1-deoxymannojirimycin. These properties are consistent with an endoplasmic reticulum-resident α1,2-mannosidase of the glycosyl hydrolase family 47. Moreover, a proteolytic activity responsible for converting the 52 kDa α-mannosidase into a polypeptide of 43 kDa retaining full enzyme activity, was demonstrated in membranes of ATCC 26555, but not in CAI-4 strain. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • 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. [ABSTRACT FROM AUTHOR]

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: Sporothrix schenckii is the causative agent of sporotrichosis. A 70-kDa glycoprotein, Gp70, is a candidate for the development of prophylactic alternatives to control the disease, and its gene (GP70) is predicted to encode for a protein of 43 kDa, contrasting with the molecular weight of the native protein., Materials & Methods: The GP70 was expressed in bacteria, the recombinant protein purified, used in immunoassays and injected to Galleria mellonella., Results & Conclusion: The recombinant protein was detected by anti-Gp70 antibodies, confirming that the Gp70 backbone is a 43-kDa peptide. This protein showed enzyme activity of cyclase and was recognized by sera of patients with sporotrichosis. Although it was not useful for serodiagnosis of sporotrichosis, it conferred protection to animals against experimental sporotrichosis.

The recognition of fungal cells by the host immune system is key during the establishment of a protective anti-fungal response. Even though the immune system has evolved a vast number of processes to control these organisms, they have developed strategies to fight back, avoiding the proper recognition by immune components and thus interfering with the host protective mechanisms. Therefore, the strategies to evade the immune system are as important as the virulence factors and attributes that damage the host tissues and cells. Here, we performed a thorough revision of the main fungal tactics to escape from the host immunosurveillance processes. These include the composition and organization of the cell wall, the fungal capsule, the formation of titan cells, biofilms, and asteroid bodies; the ability to undergo dimorphism; and the escape from nutritional immunity, extracellular traps, phagocytosis, and the action of humoral immune effectors., Competing Interests: The authors declare no conflict of interest.

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, alpha1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one alpha1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound alpha-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-alpha1,2-mannosidase antibodies. The enzyme hydrolysed Man(9)GlcNAc(2) into Man(8)GlcNAc(2) isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This alpha1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised alpha1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi alpha1,2-mannosidases and therefore, the processing of N-glycans by alpha1,2-mannosidases is similar to that present in lower eukaryotes.

Background: Interleukin (IL)-1beta has an important role in antifungal defense mechanisms. The inflammasome is thought to be required for caspase-1 activation and processing of the inactive precursor pro-IL-1beta. The aim of the present study was to investigate the pathways of IL-1beta production induced by Candida albicans in human monocytes., Methods: Human mononuclear cells were stimulated with C. albicans or mutant strains defective in mannosylation or chitin. Receptors were blocked with specific antagonists, and the IL-1beta concentration was measured., Results: Human primary monocytes produce bioactive IL-1beta when stimulated with C. albicans. The transcription of IL-1beta was induced through mannose receptor (MR), Toll-like receptor (TLR) 2, and dectin-1 but not through TLR4 and TLR9. N-mannan-linked residues, chitin, and beta-glucan from C. albicans are important for IL-1beta stimulation. Surprisingly, processing and secretion of IL-1beta in monocytes did not require pathogen-mediated inflammasome activation, because of the constitutive activation of caspase-1 and the capability of monocytes to release endogenous adenosine-5'-triphosphate., Conclusions: This study is the first dissection of the molecular mechanisms of IL-1beta production by a fungal pathogen. Transcription through mannan/chitin/MR and beta-glucan/dectin-1/TLR2 induces production of IL-1beta by C. albicans in human monocytes, whereas processing of IL-1beta is mediated by constitutively active caspase-1.

Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with alpha1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an alpha1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble alpha1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise alpha1,2-mannosidases in other biological systems as well.

The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc(3)Man(9)GlcNAc(2) N-glycan is processed by alpha-glucosidases I and II and alpha1,2-mannosidase to generate Man(8)GlcNAc(2). This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for alpha-glucosidase I, alpha-glucosidase II catalytic subunit, and alpha1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41Delta, Carot2Delta, and Camns1Delta null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated beta-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.

Beta (1,3)-glucans represent 40% of the cell wall of the yeast Candida albicans. The dectin-1 lectin-like receptor has shown to recognize fungal beta (1,3)-glucans and induce innate immune responses. The importance of beta-glucan-dectin-1 pathways for the recognition of C. albicans by human primary blood cells has not been firmly established. In this study we demonstrate that cytokine production by both human peripheral blood mononuclear cells and murine macrophages is dependent on the recognition of beta-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of beta-glucans on the surface of the cell wall and subsequent recognition by dectin-1, whereas live yeasts stimulated monocytes mainly via recognition of cell-surface mannans. Dectin-1 induced cytokine production through the following 2 pathways: Syk-dependent production of the T-helper (Th) 2-type anti-inflammatory cytokine interleukin-10 and Toll-like receptor-Myd88-dependent stimulation of monocyte-derived proinflammatory cytokines, such as tumor necrosis factor-alpha . In contrast, stimulation of Th1-type cytokines, such as interferon-gamma , by C. albicans was independent of the recognition of beta-glucans by dectin-1. In conclusion, C. albicans induces production of monocyte-derived and T cell-derived cytokines through distinct pathways dependent on or independent of dectin-1.