Search Results (24)

Background/Objectives: The mycobacterial complex cell envelope serves as a formidable barrier against host immunity and antibiotics. Lipomannan (LM) and lipoarabinomannan (LAM) are key structural components of the mycobacterial envelope and potent immunomodulators. The mycobacterial lipoarabinomannan biosynthesis mannosyltransferase MptC modifies the multiple α-(1→2)-linked branched mannan residues of LAM in the mycobacteria. However, the role of MptC in mycobacterial infectivity, antibiotic susceptibility and host immune regulation remains poorly understood. Methods: An mptC (also named MSMEG_4247) knockout Mycobacterium smegmatis mc²-155 (M. smeg) strain (designated as M. smegΔmptC) was generated using CRISPR–Cas12a technology. The effects of MptC on bacterial physiology, cell wall permeability, drug sensitivity, immune cell function, and survival during infection are analyzed through glycogen staining, drug sensitivity tests, and cellular and mouse infection models. Results: MptC deficiency results in a loss of LM and increase in LAM synthesis. The M. smegΔmptC mutant strain exhibits enhanced cell wall permeability and reduces hydrophobicity. Functionally, the mptC knockout strain increases the intracellular cytokines (IFN-γ, TNF-a and IL-17) production of T cells in mice. Consequently, results based on both macrophage and mouse infection models demonstrate that the M. smegΔmptC strain has less bacterial loads and higher susceptibility to antibiotic rifampicin. Conclusions: Mannosyltransferase MptC plays an important role in maintaining cell wall integrity (via LM/LAM synthesis), regulating T cell responses, and influencing antibiotic susceptibility in mycobacteria. Full article

Mycobacterium tuberculosis, the causative agent of tuberculosis, utilizes biofilm formation as a key mechanism to withstand host-derived stresses. To identify novel factors involved in this process, we performed a CRISPRi screen in the model organism Mycobacterium smegmatis. This screen identified trypsin HtrA as a critical factor for growth and biofilm formation. Deletion of htrA led to a profound upregulation of the cell wall amidase Ami3. We demonstrated that Ami3 is a crucial negative regulator of biofilm formation, as overexpression of ami3 recapitulated the biofilm and growth defects of the ΔhtrA strain. Furthermore, we found that the essential role of periplasmic protease HtrA for normal growth could be suppressed by novel mutations in pmt, a gene encoding a phosphomyoinositol mannosyltransferase, at residues F53 and N55, distinct from the previously reported D68 site. Our findings establish a novel regulatory pathway in which HtrA modulates mycobacterial biofilm formation by controlling the levels of Ami3 and reveal new genetic interactions within this network. Full article

The global rise in resistance to first-line antifungal agents highlights the urgent need for new therapeutic strategies. Glycosylphosphatidylinositol (GPI)-anchored protein biosynthesis is an attractive target. The GPI mannosyltransferase I (GPI-MT-I), composed of Gpi14 and Pbn1, catalyzes the essential first mannose transfer from dolichol-phosphomannose (Dol-P-Man) to the GPI precursor. This initial mannosylation is critical for fungal cell wall integrity, yet the molecular basis of GPI-MT-I assembly and substrate recognition remains poorly understood. Here, we present the cryo-EM structure of Candida glabrata GPI-MT-I in complex with Dol-P-Man, revealing how Gpi14 and Pbn1 form a stable complex and engage the mannose donor. An AlphaFold3-predicted acceptor-bound model further defines the structural basis of acceptor substrate recognition and suggests a plausible catalytic mechanism. Comparison with structural homologs highlights a distinct mode of substrate engagement by GPI-MT-I. Together, these findings establish a mechanistic framework for GPI-MT-I function with broader implications for the GPI-MT family. Full article

Rice false smut, caused by Ustilaginoidea virens, is one of the three major rice diseases in China. It not only seriously affects the rice yield and quality but also endangers human and animal health. Studying the pathogenic mechanism of U. virens has important theoretical significance and application value for clarifying the infection characteristics of the pathogen and cultivating disease-resistant varieties. Plant pathogenic fungi utilize secreted effectors to suppress plant immune responses, which can function in the apoplast or within host cells and are likely glycosylated. However, the posttranslational regulation of these effectors remains unexplored. Deletion of ΔUvALG led to the cessation of secondary infection hyphae growth and a notable decrease in virulence. We observed that ΔUvALG mutants triggered a significant increase in reactive species production within host cells, akin to ALG mutants, which plays a crucial role in halting the growth of infection hyphae in the mutants. ALG functions by sequestering chitin oligosaccharides to prevent their recognition by the rice chitin elicitor, thereby inhibiting the activation of innate immune responses, including reactive species production. Our findings reveal that ALG3 possesses three N-glycosylation sites, and the simultaneous Alg-mediated N-glycosylation of each site is essential for maintaining protein stability and chitin-binding activity, both of which are critical for its effector function. These outcomes underscore the necessity of the Alg-mediated N-glycosylation of ALG to evade host innate immunity. Full article

Camellia oleifera is a woody, edible-oil plant native to China. Anthracnose is the major disease of Ca. oleifera, and Colletotrichum fructicola is the main epidemic pathogen. Our previous research indicated that CfHac1 (homologous to ATF/CREB1) and CfGcn5 (general control nonderepressible 5, Gcn5) are integral to key cellular processes that govern fungal development and pathogenesis. Further transcriptomic analyses of the CfHac1 and CfGcn5 mutants, particularly under conditions of endoplasmic reticulum (ER) stress, hold the potential to unveil additional genes implicated in this critical cellular response. We identified all OST/PMT (oligosaccharyltransferase/Protein O-Mannosyltransferases) genes in C. fructicola and analyzed their expression levels. To elucidate novel glycosylation-related genes that may be important for the virulence of C. fructicola, we took an unbiased transcriptomic approach comparing wild-type and the ∆Cfhac1 mutant. Notably, all OST/PMT genes were induced by dithiothreitol and down-regulated in the ΔCfhac1 mutant, yet only the CfPMT4 (Protein O-Mannosyltransferases 4) gene (A04626) was unaffected in the ΔCfgcn5. The results of targeted gene deletion experiments indicate that CfPMT4 plays a crucial role in both vegetative growth and conidiation. Additionally, our investigation revealed that the ΔCfpmt4 exhibits deficiencies in appressorium formation, as well as in its response to cell wall integrity and endoplasmic reticulum stresses. Furthermore, the mutant displayed impaired glycogen metabolism, which may contribute to reduced penetration ability. Overall, CfPmt4, an O-mannosyltransferase, controls the growth, development, and pathogenicity of Colletotrichum fructicola. Understanding the function of the CfPMT4 homolog could provide a potential molecular target for controlling Ca. oleifera anthracnose. Full article

α-1,2-mannosyltransferase (ALG9) germline variants are linked to autosomal dominant polycystic kidney disease (ADPKD). Many individuals affected with ADPKD possess polycystic livers as a common extrarenal manifestation. We performed whole exome sequencing in a female with autosomal dominant polycystic liver disease (ADPLD) without kidney cysts and established the presence of a heterozygous missense variant (c.677G>C p.(Gly226Ala)) in ALG9. In silico pathogenicity prediction and 3D protein modeling determined this variant as pathogenic. Loss of heterozygosity is regularly seen in liver cyst walls. Immunohistochemistry indicated the absence of ALG9 in liver tissue from this patient. ALG9 expression was absent in cyst wall lining from ALG9- and PRKCSH-caused ADPLD patients but present in the liver cyst lining derived from an ADPKD patient with a PKD2 variant. Thus, heterozygous pathogenic variants in ALG9 are also associated with ADPLD. Somatic loss of heterozygosity of the ALG9 enzyme was seen in the ALG9 patient but also in ADPLD patients with a different genetic background. This expanded the phenotypic spectrum of ADPLD to ALG9. Full article

Earlier studies have shown that the outer layers of the conidial and mycelial cell walls of Aspergillus fumigatus are different. In this work, we analyzed the polysaccharidome of the resting conidial cell wall and observed major differences within the mycelium cell wall. Mainly, the conidia cell wall was characterized by (i) a smaller amount of α-(1,3)-glucan and chitin; (ii) a larger amount of β-(1,3)-glucan, which was divided into alkali-insoluble and water-soluble fractions, and (iii) the existence of a specific mannan with side chains containing galactopyranose, glucose, and N-acetylglucosamine residues. An analysis of A. fumigatus cell wall gene mutants suggested that members of the fungal GH-72 transglycosylase family play a crucial role in the conidia cell wall β-(1,3)-glucan organization and that α-(1,6)-mannosyltransferases of GT-32 and GT-62 families are essential to the polymerization of the conidium-associated cell wall mannan. This specific mannan and the well-known galactomannan follow two independent biosynthetic pathways. Full article

Mutations in the extracellular matrix protein eyes shut homolog (EYS) are a common cause of retinitis pigmentosa, a blinding disease characterized by photoreceptor degeneration. EYS binds to matriglycan, a carbohydrate modification on O-mannosyl glycan substitutions of the cell-surface glycoprotein α-dystroglycan. Patients with mutations in enzymes required for the biosynthesis of matriglycan exhibit syndromic retinal atrophy, along with brain malformations and congenital muscular dystrophy. Protein O-mannosyltransferase 2 (POMT2) is an enzyme required for the synthesis of O-mannosyl glycans. To evaluate the roles of O-mannosyl glycans in photoreceptor health, we generated protein O-mannosyltransferase 2 (pomt2) mutant zebrafish by CRISPR. pomt2 mutation resulted in a loss of matriglycan and abolished binding of EYS protein to α-dystroglycan. Mutant zebrafish presented with hydrocephalus and hypoplasia of the cerebellum, as well as muscular dystrophy. EYS protein was enriched near photoreceptor connecting cilia in the wild-type, but its presence and proper localization was significantly reduced in mutant animals. The mutant retina exhibited mis-localization of opsins and increased apoptosis in both rod and cone photoreceptors. Immunofluorescence intensity of G protein subunit alpha transducin 2 (GNAT2) antibody (a general cone marker) and 1D4 antibody (a long double cone marker) in mutant retinas did not differ from wild-type retinas at 1-month post fertilization, but was reduced at 6 months post fertilization, indicating significant cone degeneration. These data suggest that POMT2-mediated O-mannosyl glycosylation is required for EYS protein localization to the connecting cilium region and photoreceptor survival. Full article

Peritoneal metastasis is the main cause of poor prognoses and high mortality in ovarian cancer patients. Abnormal protein glycosylation modification is associated with cancer malignancy. Elevated α1,3-mannosyltransferase 3 (ALG3), which catalyzes the α1,3-mannosylation of glycoproteins, has been found in some malignant tumors. However, the pathological significance of ALG3 and its regulatory mechanism in ovarian cancer metastasis is unclear. The results showed that the level of ALG3/α1,3-mannosylation was higher in human ovarian cancer tissues compared with normal ovarian tissues, as measured by Lectin chip, Western blot and Lectin blot analyses, as well as ovarian tissue microarray analysis. ALG3 was also correlated with the poor prognosis of ovarian cancer patients, according to survival analysis. The downregulation of ALG3 decreased the proliferation, stemness and peritoneal metastasis of ovarian cancer cells. The increase in urokinase plasminogen activator receptor (uPAR) α1,3-mannosylation catalyzed by ALG3 enhanced urokinase plasminogen activator (uPA)/uPAR activation and the interaction of uPAR with a disintegrin and metalloproteinase 8 (ADAM8), which promoted ovarian cancer peritoneal metastasis via the ADAM8/Ras/ERK pathway. Furthermore, decreased ALG3 suppressed ascites formation and the peritoneal metastasis of ovarian cancer cells in mice. This study highlights ALG3 as a potential diagnostic biomarker and prospective therapeutic target for ovarian cancer. Full article

Glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is an essential glycosyltransferase of glycosylphosphatidylinositol-anchor proteins (GPI-APs) that transfers the first of the four mannoses in GPI-AP precursors, which have multiple functions, including immune response and signal transduction. In this study, the GPI-MT-I gene that regulates GPI-AP biosynthesis in Andrias davidianus (AdGPI-MT-I) was characterized for the first time. The open reading frame (ORF) of AdGPI-MT-I is 1293 bp and encodes a protein of 430 amino acids that contains a conserved PMT2 superfamily domain. AdGPI-MT-I mRNA was widely expressed in the tissues of the Chinese giant salamander. The mRNA expression level of AdGPI-MT-I in the spleen, kidney, and muscle cell line (GSM cells) was significantly upregulated post Chinese giant salamander iridovirus (GSIV) infection. The mRNA expression of the virus major capsid protein (MCP) in AdGPI-MT-I-overexpressed cells was significantly reduced. Moreover, a lower level of virus MCP synthesis and gene copying in AdGPI-MT-I-overexpressed cells was confirmed by western blot and ddPCR. These results collectively suggest that GSIV replication in GSM cells was significantly reduced by the overexpression of the AdGPI-MT-I protein, which may contribute to a better understanding of the antiviral mechanism against iridovirus infection. Full article

The hard-to-cook defect in common beans is dictated by the ability to achieve cell separation during cooking. Hydrolysis of pectin methyl-esters by the pectin methyl-esterase (PME) enzyme influences cell separation. However, the contributions of the PME enzyme and the cell wall to the hard-to-cook defect have not been studied using molecular tools. We compared relevant molecular processes in fast- and slow-cooking bean varieties to understand the mechanisms underpinning the hard-to-cook defect. A PME spectrophotometric assay showed minor differences in enzyme activity between varieties. Meanwhile, a PME HMMER search in the P. vulgaris genome unveiled 113 genes encoding PMEs and PME inhibitors (PMEIs). Through RNA sequencing, we compared the gene expression of the PME-related genes in both varieties during seed development. A PME (Phvul010g080300) and PMEI gene (Phvul005g007600) showed the highest expression in the fast- and slow-cooking beans, respectively. We further identified 2132 differentially expressed genes (DEGs). Genes encoding cell-wall-related enzymes, mainly glycosylphosphatidylinositol mannosyltransferase, xyloglucan O-acetyltransferase, pectinesterase, and callose synthase, ranked among the top DEGs, indicating novel relations to the hard-to-cook defect. Gene ontology mapping revealed hydrolase activity and protein phosphorylation as functional categories with the most abundant upregulated DEGs in the slow-cooking bean. Additionally, the cell periphery contained 8% of the DEGs upregulated in the slow-cooking bean. This study provides new insights into the role of pectin methyl-esterase-related genes and novel cell wall processes in the occurrence of the hard-to-cook defect. Full article

Redundancy and lethality is a long-standing problem in genetics but generating minimal and lethal phenotypes in the knockouts of the same gene by different approaches drives this problem to a new high. In Asn (N)-linked glycosylation, a complex and ubiquitous cotranslational and post-translational protein modification required for the transfer of correctly folded proteins and endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, ALG12 (EBS4) is an α 1, 6-mannosyltransferase catalyzing a mannose into Glc₃Man₉GlcNAc₂. In Arabidopsis, T-DNA knockout alg12-T is lethal while likely ebs4 null mutants isolated by forward genetics are most healthy as weak alleles, perplexing researchers and demanding further investigations. Here, we isolated a true null allele, sbi2, with the W258Stop mutation in ALG12/EBS4. sbi2 restored the sensitivity of brassinosteroid receptor mutants bri1-5, bri1-9, and bri1-235 with ER-trapped BRI1 to brassinosteroids. Furthermore, sbi2 maturated earlier than the wild-type. Moreover, concomitant with impaired and misfolded proteins accumulated in the ER, sbi2 had higher sensitivity to tunicamycin and salt than the wild-type. Our findings thus clarify the role of SBI2/ALG12/EBS4 in the regulation of the ERAD of misfolded glycoproteins, and plant growth and stress response. Further, our study advocates the necessity and importance of using multiple approaches to validate genetics study. Full article

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation. Full article

C-Mannosylation is a post-translational modification of proteins in the endoplasmic reticulum. Monomeric α-mannose is attached to specific Trp residues at the first Trp in the Trp-x-x-Trp/Cys (W-x-x-W/C) motif of substrate proteins, by the action of C-mannosyltransferases, DPY19-related gene products. The acceptor substrate proteins are included in the thrombospondin type I repeat (TSR) superfamily, cytokine receptor type I family, and others. Previous studies demonstrated that C-mannosylation plays critical roles in the folding, sorting, and/or secretion of substrate proteins. A C-mannosylation-defective gene mutation was identified in humans as the disease-associated variant affecting a C-mannosylation motif of W-x-x-W of ADAMTSL1, which suggests the involvement of defects in protein C-mannosylation in human diseases such as developmental glaucoma, myopia, and/or retinal defects. On the other hand, monomeric C-mannosyl Trp (C-Man-Trp), a deduced degradation product of C-mannosylated proteins, occurs in cells and extracellular fluids. Several studies showed that the level of C-Man-Trp is upregulated in blood of patients with renal dysfunction, suggesting that the metabolism of C-Man-Trp may be involved in human kidney diseases. Together, protein C-mannosylation is considered to play important roles in the biosynthesis and functions of substrate proteins, and the altered regulation of protein C-manosylation may be involved in the pathophysiology of human diseases. In this review, we consider the biochemical and biomedical knowledge of protein C-mannosylation and C-Man-Trp, and introduce recent studies concerning their significance in biology and medicine. Full article

Protein O-mannosyltransferases (Pmts) comprise a group of proteins that add mannoses to substrate proteins at the endoplasmic reticulum. This post-translational modification is important for the faithful transfer of nascent glycoproteins throughout the secretory pathway. Most fungi genomes encode three O-mannosyltransferases, usually named Pmt1, Pmt2, and Pmt4. In pathogenic fungi, Pmts, especially Pmt4, are key factors for virulence. Although the importance of Pmts for fungal pathogenesis is well established in a wide range of pathogens, questions remain regarding certain features of Pmts. For example, why does the single deletion of each pmt gene have an asymmetrical impact on host colonization? Here, we analyse the origin of Pmts in fungi and review the most important phenotypes associated with Pmt mutants in pathogenic fungi. Hence, we highlight the enormous relevance of these glycotransferases for fungal pathogenic development. Full article