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Comprehensive phenotypic analysis of multiple gene deletions of α-glucan synthase and Crh-transglycosylase gene families in Aspergillus niger highlighting the versatility of the fungal cell wall

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2025-01-31 DOI: 10.1016/j.tcsw.2025.100141

Katharina J. Ost , Mark Arentshorst , Bruno M. Moerschbacher , Mareike E. Dirks-Hofmeister , Arthur F.J. Ram

Multiple paralogs are found in the fungal genomes for several genes that encode proteins involved in cell wall biosynthesis. The genome of A. niger contains five genes encoding putative α-1,3-glucan synthases (AgsA-E) and seven genes encoding putative glucan-chitin crosslinking enzymes (CrhA-G). Here, we systematically studied the effects of the deletion of single (agsA or agsE), double (agsA and agsE), or all five ags genes (agsA-E) present in A. niger. Morphological and biochemical analysis of ags mutants emphasizes the important role of agsE in cell wall integrity, while deletion of other ags genes had minimal impact. Loss of agsE compromised cell wall integrity and altered pellet morphology in liquid cultures.

Previous studies have indicated that deletion of all crh genes in A. niger did not result in cell wall integrity-related phenotypes. To determine whether the ags and crh gene families have redundant functions, both gene families were deleted using iterative CRISPR/Cas9 mediated genome editing. The 12-fold deletion mutant was viable and did not exhibit growth defects under non-stressing growth conditions. A synergistic effect on cell wall integrity was observed in this 12-fold deletion mutant, particularly when exposed to cell wall-perturbing compounds. The cell wall composition, extractability of glucans by alkali, and scanning electron microscopy analysis showed no differences between the parental strain and mutants lacking ags genes, crh genes, or both. These observations underscore the ability of fungal cells to adapt and secure cell wall integrity, even when two entire cell wall protein-encoding gene families are missing.

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引用次数: 0

Sporothrix brasiliensis Gp70 is a cell wall protein required for adhesion, proper interaction with innate immune cells, and virulence

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2025-01-06 DOI: 10.1016/j.tcsw.2024.100139

Leonardo Padró-Villegas , Manuela Gómez-Gaviria , Iván Martínez-Duncker , Luz A. López-Ramírez , José A. Martínez-Álvarez , Gustavo A. Niño-Vega , Héctor M. Mora-Montes

Sporothrix brasiliensis is one of the leading etiological agents of sporotrichosis, a cutaneous and subcutaneous mycosis worldwide distributed. This organism has been recently associated with epidemic outbreaks in Brazil. Despite the medical relevance of this species, little is known about its virulence factors, and most of the information on this subject is extrapolated from Sporothrix schenckii. Here, we generated S. brasiliensis mutants, where GP70 was silenced. In S. schenckii, this gene encodes a glycoprotein with adhesive properties required for virulence. The S. brasiliensis GP70 silencing led to an abnormal cellular phenotype, with smaller, round yeast-like cells that aggregate. Cell aggregation was disrupted with glucanase, suggesting this phenotype is linked to changes in the cell wall. The cell wall characterization confirmed changes in the structural polysaccharide β-1,3-glucan, which increased in quantity and exposure at the cell surface. This was accompanied by a reduction in protein content and N-linked glycans. Mutant strains with high GP70-silencing levels showed minimal levels of 3-carboxy-cis,cis-muconate cyclase activity, this glycoprotein's predicted enzyme function, and decreased ability to bind laminin and fibronectin. These phenotypical changes coincided with abnormal interaction with human peripheral blood mononuclear cells, where production of IL-1β, IL-17, and IL-22 was reduced and the strong dependence on cytokine stimulation via mannose receptor was lost. Phagocytosis by monocyte-derived macrophages was increased and virulence attenuated in a Galleria mellonella larvae. In conclusion, Gp70 is an abundant cell wall glycoprotein in S. brasiliensis that contributes to virulence and proper interaction with innate immnune cells.

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引用次数: 0

Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2025-01-03 DOI: 10.1016/j.tcsw.2024.100140

Apurva Chatrath, Pavan Patel, Protyusha Dey, Stephen J. Free

In Neurospora crassa vegetative hyphae, chitin, β-1,3-glucan (laminarin), and a mixed β-1,3−/β-1,4-glucan (lichenin) are the major cell wall polysaccharides. GH72 enzymes have been shown to function as β-1,3-glucanases and glucanosyltransferases and can function in crosslinking β-1,3-glucans together. To characterize the enzymatic activities of the N. crassa enzymes, we expressed GEL-1 with a HIS6 tag in N. crassa. A chimeric maltose binding protein:GEL-2 was produced in E. coli. Purified GEL-1 and GEL-2 were used to characterize their enzymatic activities. We employed thin-layer chromatography (TLC) and polyacrylamide carbohydrate gel electrophoresis (PACE) assays to visualize GEL-1 and GEL-2 hydrolase and transferase activities on lichenin and laminarin substrates. We determined that GEL-1 functions as a laminarinase (β-1,3-glucanase) and as a laminarin transferase. We found that GEL-2 can function as a laminarinase and as a licheninase (β-1,3−/β-1,4-mixed-glucanase) and can crosslink β-1,3-glucans together. We demonstrated that GEL-2 can form enzyme:lichenin intermediates, providing evidence that GEL-2 functions as a lichenin transferase as well as a β-1,3-glucan transferase and crosslinks both types of polysaccharides into the N. crassa cell wall.

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引用次数: 0

Endocytic tethers modulate unconventional GAPDH secretion 内吞系索调节非常规GAPDH分泌。

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2024-12-24 DOI: 10.1016/j.tcsw.2024.100138

Michael J. Cohen , Brianne Philippe , Peter N. Lipke

Yeast cell walls contain both classically-secreted and unconventionally-secreted proteins. The latter class lacks the signal sequence for translocation into the ER, therefore these proteins are transported to the wall by uncharacterized mechanisms. One such protein is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is abundant in the cytosol, but also found in the yeast cell wall where it is enzymatically active. We screened diploid Saccharomyces cerevisiae homozygous gene deletions for changes in cell wall GAPDH activity. Deletions targeting endocytic tethers in the endolysosomal system had the largest effects on GAPDH secretion, including vps21, bro1, vps41, and pep12. The predominant GAPDH isoform Tdh3 was partially localized to endolysosomal compartments, including multivesicular bodies, which are common entry points to unconventional protein secretion pathways. Yeast lacking the endosomal Rab5-GTPase Vps21 had defects in GAPDH secretion as well as delayed entry into to the endolysosomal compartments. Therefore, we conclude that entry into the endolysosomal compartment facilitates non-conventional secretion of GAPDH.

酵母细胞壁包含经典分泌和非常规分泌的蛋白质。后一类缺乏转运到内质网的信号序列，因此这些蛋白质通过未知的机制转运到壁。其中一种蛋白质是糖酵解酶甘油醛-3-磷酸脱氢酶（GAPDH），它丰富于细胞质中，但也存在于酵母菌细胞壁中，在那里它具有酶活性。我们筛选二倍体酿酒酵母纯合基因缺失的细胞壁GAPDH活性的变化。内溶酶体系统中针对内噬系链的缺失对GAPDH分泌的影响最大，包括vps21、bro1、vps41和pep12。主要的GAPDH亚型Tdh3部分定位于内溶酶体室室，包括多泡体，这是非常规蛋白质分泌途径的常见入口点。缺乏内体Rab5-GTPase Vps21的酵母在GAPDH分泌方面存在缺陷，并且延迟进入内溶酶体腔室。因此，我们得出结论，进入内溶酶体室有助于GAPDH的非常规分泌。

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引用次数: 0

Mucilicious methods: Navigating the tools developed to Arabidopsis Seed Coat Mucilage analysis 黏液分析方法：导航拟南芥种皮黏液分析工具。

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2024-12-11 DOI: 10.1016/j.tcsw.2024.100134

Susana Saez-Aguayo , Dayan Sanhueza , Vicente Jara , Benjamin Galleguillos , Alfonso Gonzalo de la Rubia , Asier Largo-Gosens , Adrian Moreno

During the last decades, Arabidopsis seed mucilage has been extensively studied to gain insight into the metabolism of pectin, hemicellulose and cellulose. This review aims to provide a comprehensive examination of the techniques used to understand the composition and structure of Arabidopsis mucilage. Moreover, we present novel findings from mucilage analysis, including the separation of pectic domains within the mucilage, offering a fresh perspective on utilizing traditional techniques to analyze mucilage mutant lines.

在过去的几十年里，人们对拟南芥种子粘液进行了广泛的研究，以深入了解果胶、半纤维素和纤维素的代谢。本文综述了拟南芥粘液组成和结构的研究进展。此外，我们还介绍了粘液分析的新发现，包括粘液中果胶结构域的分离，为利用传统技术分析粘液突变系提供了新的视角。

引用次数: 0

Staphylococcus aureus: Current perspectives on molecular pathogenesis and virulence 金黄色葡萄球菌：分子发病机制和毒力的最新研究进展。

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2024-12-09 DOI: 10.1016/j.tcsw.2024.100137

Abayeneh Girma

Staphylococcus aureus has evolved a sophisticated regulatory system to control its virulence. One of the main roles of this interconnected network is to sense and respond to diverse environmental signals by altering the synthesis of virulence components required for survival in the host, including cell surface adhesins, extracellular enzymes and toxins. The accessory gene regulator (agr), a quorum sensing system that detects the local concentration of a cyclic peptide signaling molecule, is one of the well-studied of these S. aureus regulatory mechanisms. By using this system, S. aureus is able to sense its own population density and translate this information into a specific pattern of gene expression. In addition to Agr, this pathogen senses specific stimuli through various two-component systems and synchronizes responses with alternative sigma factors and cytoplasmic regulators of the SarA protein family. These different regulatory mechanisms combine host and environmental information into a network that guarantees the best possible response of pathogens to changing circumstances. In this article, an overview of the most significant and thoroughly studied regulatory systems of S. aureus is provided, along with a summary of their roles in host interactions.

金黄色葡萄球菌已经进化出一个复杂的调控系统来控制它的毒力。这个相互连接的网络的主要作用之一是通过改变宿主生存所需的毒力成分的合成，包括细胞表面粘附素、细胞外酶和毒素，来感知和响应各种环境信号。辅助基因调控（agr）是一种群体感应系统，可检测环肽信号分子的局部浓度，是这些金黄色葡萄球菌调控机制中研究得很好的一个。通过使用这个系统，金黄色葡萄球菌能够感知自身的种群密度，并将这些信息转化为特定的基因表达模式。除了Agr，这种病原体通过各种双组分系统感知特定刺激，并与其他sigma因子和SarA蛋白家族的细胞质调节因子同步反应。这些不同的调节机制将宿主和环境信息结合成一个网络，保证病原体对不断变化的环境做出最佳反应。本文概述了金黄色葡萄球菌最重要和最深入研究的调控系统，并总结了它们在宿主相互作用中的作用。

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引用次数: 0

Spatiotemporal regulation of alginate sub-structures at multiple scales revealed by monoclonal antibodies

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2024-12-08 DOI: 10.1016/j.tcsw.2024.100136

Catherine T. Jones , Cassie Bakshani , Ieva Lelenaite , Jozef Mravec , Stjepan Krešimir Kračun , Jeff Pearson , Mathew D. Wilcox , Kevin Hardouin , Sonia Kridi , Cécile Hervé , William G.T. Willats

Alginates are abundant linear polysaccharides produced by brown algae and some bacteria. They have multiple biological roles and important medical and commercial uses. Alginates are comprised of D-mannuronic acid (M) and L-guluronic acid (G) and the ratios and distribution patterns of M and G profoundly impact their physiological and rheological properties. The structure/function relationships of alginates have been extensively studied in vitro but our understanding of the in vivo spatiotemporal regulation of alginate fine structures and their biological implications is limited. Monoclonal antibodies (mAbs) are powerful tools for localising and quantifying glycan structures and several alginate-directed mAbs have been developed. We used a library of well-defined alginates, with M and G block ratios determined by NMR, to refine our understanding of the binding properties of alginate-directed mAbs. Using these probes, we obtained new insight into how structural features of alginates are regulated at different scales, from cellular to seasonal.

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引用次数: 0

Antimicrobial peptides and proteins against drug-resistant pathogens 抗耐药性病原体的抗菌肽和蛋白质

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2024-12-01 DOI: 10.1016/j.tcsw.2024.100135

Yeji Wang, Minghui Song, Wenqiang Chang

The rise of drug-resistant pathogens, driven by the misuse and overuse of antibiotics, has created a formidable challenge for global public health. Antimicrobial peptides and proteins have garnered considerable attention as promising candidates for novel antimicrobial agents. These bioactive molecules, whether derived from natural sources, designed synthetically, or predicted using artificial intelligence, can induce lethal effects on pathogens by targeting key microbial structures or functional components, such as cell membranes, cell walls, biofilms, and intracellular components. Additionally, they may enhance overall immune defenses by modulating innate or adaptive immune responses in the host. Of course, development of antimicrobial peptides and proteins also face some limitations, including high toxicity, lack of selectivity, insufficient stability, and potential immunogenicity. Despite these challenges, they remain a valuable resource in the fight against drug-resistant pathogens. Future research should focus on overcoming these limitations to fully realize the therapeutic potential of antimicrobial peptides in the infection control.

抗生素的滥用和过度使用导致耐药病原体的增加，给全球公共卫生带来了巨大挑战。抗菌肽和蛋白质作为新型抗菌药物的有前途的候选者已经引起了相当大的关注。这些生物活性分子，无论是来自天然来源、合成设计还是使用人工智能预测，都可以通过靶向关键微生物结构或功能成分（如细胞膜、细胞壁、生物膜和细胞内成分）对病原体产生致命影响。此外，它们可能通过调节宿主的先天或适应性免疫反应来增强整体免疫防御。当然，抗菌肽和抗菌蛋白的发展也面临着一些限制，包括高毒性、缺乏选择性、稳定性不足和潜在的免疫原性。尽管存在这些挑战，它们仍然是抗击耐药病原体的宝贵资源。今后的研究应着眼于克服这些局限性，以充分发挥抗菌肽在感染控制中的治疗潜力。

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引用次数: 0

Advances on cell wall biology: Highlights from the XVI Plant Cell Wall Meeting 细胞壁生物学研究进展：第十六届植物细胞壁会议要点

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2024-12-01 DOI: 10.1016/j.tcsw.2024.100130

Hugo Mélida , Antonio Molina

引用次数: 0

Combatting biofilm-mediated infections in clinical settings by targeting quorum sensing 以法定人数感应为目标，抗击临床环境中生物膜介导的感染

Q1 Immunology and Microbiology

Cell Surface

Pub Date : 2024-11-08 DOI: 10.1016/j.tcsw.2024.100133

Arindam Mitra

Biofilm-associated infections constitute a significant challenge in managing infectious diseases due to their high resistance to antibiotics and host immune responses. Biofilms are responsible for various infections, including urinary tract infections, cystic fibrosis, dental plaque, bone infections, and chronic wounds. Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to coordinate gene expression in response to cell density, which is crucial for biofilm formation and maintenance.. Its disruption has been proposed as a potential strategy to prevent or treat biofilm-associated infections leading to improved treatment outcomes for infectious diseases. This review article aims to provide a comprehensive overview of the literature on QS-mediated disruption of biofilms for treating infectious diseases. It will discuss the mechanisms of QS disruption and the various approaches that have been developed to disrupt QS in reference to multiple clinical pathogens. In particular, numerous studies have demonstrated the efficacy of QS disruption in reducing biofilm formation in various pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus. Finally, the review will discuss the challenges and future directions for developing QS disruption as a clinical therapy for biofilm-associated infections. This includes the development of effective delivery systems and the identification of suitable targets for QS disruption. Overall, the literature suggests that QS disruption is a promising alternative to traditional antibiotic treatment for biofilm-associated infections and warrants further investigation.

由于生物膜对抗生素和宿主免疫反应具有很强的抗药性，因此与生物膜相关的感染是控制传染病的一大挑战。生物膜是各种感染的罪魁祸首，包括尿路感染、囊性纤维化、牙菌斑、骨感染和慢性伤口。法定量感应（QS）是细菌根据细胞密度协调基因表达的细胞间通信过程，对生物膜的形成和维持至关重要。人们已提出将破坏这一过程作为预防或治疗生物膜相关感染的潜在策略，从而改善传染性疾病的治疗效果。这篇综述文章旨在全面概述有关 QS 介导的破坏生物膜以治疗传染性疾病的文献。文章将讨论 QS 破坏的机制以及针对多种临床病原体开发的各种破坏 QS 的方法。特别是，许多研究已经证明了 QS 破坏在减少包括铜绿假单胞菌和金黄色葡萄球菌在内的各种病原体的生物膜形成方面的功效。最后，本综述将讨论开发 QS 破坏作为生物膜相关感染临床疗法所面临的挑战和未来发展方向。这包括开发有效的给药系统和确定合适的 QS 破坏靶点。总之，文献表明 QS 干扰是治疗生物膜相关感染的传统抗生素疗法的一种有前途的替代疗法，值得进一步研究。

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