Cell Surface最新文献

Cell walls are essential in the interaction of fungi with the (a)biotic environment and are also key to hyphal morphogenesis and mechanical strength. Here, we used solid-state NMR (ssNMR) spectroscopy combined with HPLC and GC–MS to study the structural organization of the cell wall of a representative of the Basidiomycota, one of the two main phyla of fungi. Based on the data we propose a refined model for the cell wall of a basidiomycete. In this model, the rigid core is built from α- and β-(1,3)-glucan, β-(1,3)-(1,6)-glucan, highly branched and single stranded β-(1,4)-chitin as well as polymeric fucose. The mobile fraction of the cell wall is composed of β-(1,3)-glucan, β-(1,3)-(1,6)-glucan, β-(1,6)-glucan, α-linked reducing and non-reducing ends and polymeric mannose. Together, these findings provide novel insights into the structural organization of the cell wall of the model basidiomycete S. commune that was previously based on destructive chemical and enzymatic analysis.

We designed experiments to assess whether fungal cell wall mannans function as an immune shield or an immune agonist. Fungal cell wall β-(1,3)-glucan normally plays a major and dominant role in immune activation. The outer mannan layer has been variously described as an immune shield, because it has the potential to mask the underlying β-(1,3)-glucan, or an immune activator, as it also has the potential to engage with a wide range of mannose detecting PRRs. To resolve this conundrum we examined species-specific differences in host immune recognition in the och1Δ N-mannosylation-deficient mutant background in four species of yeast-like fungi. Irrespective of the fungal species, the cytokine response (TNFα and IL-6) induced by the och1Δ mutants in human monocytes was reduced compared to that of the wild type. In contrast, TNFα production induced by och1Δ was increased, relative to wild type, due to increased β-glucan exposure, when mouse or human macrophages were used. These observations suggest that N-mannan is not a major PAMP for macrophages and that in these cells mannan does shield the fungus from recognition of the inner cell wall β-glucan. However, N-mannan is a significant inducer of cytokine for monocytes. Therefore the metaphor of the fungal “mannan shield” can only be applied to some, but not all, myeloid cells used in immune profiling experiments of fungal species.

The emergence and perseverance of drug resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of tuberculosis research. There are numerous different approaches that can be employed to lead to the discovery of anti-tubercular agents. In this work, we endeavored to optimize the anthraquinone chemical scaffold of a known drug, rhein, converting it from a compound with negligible activity against Mtb, to a series of compounds with potent activity. Two compounds exhibited low toxicity and good liver microsome stability and were further progressed in attempts to identify the biological target. Whole genome sequencing of resistant isolates revealed inactivating mutations in a monoglyceride lipase. Over-expression trials and an enzyme assay confirmed that the designed compounds are prodrugs, activated by the monoglyceride lipase. We propose that rhein is the active moiety of the novel compounds, which requires chemical modifications to enable access to the cell through the extensive cell wall structure. This work demonstrates that re-engineering of existing antimicrobial agents is a valid method in the development of new anti-tubercular compounds.

Objectives

The search for new TB drugs has become one of the great challenges for modern medicinal chemistry. An improvement in the outcomes of TB chemotherapy can be achieved by the development of new, shorter, cheap, safe and effective anti-TB regimens.

Methods

Chalcones (1a-1o) were synthesized and evaluated for their antimycobacterial activity against Mycobacterium bovis BCG using growth inhibition assays. Compound 1a was selected as a ‘hit’ compound. The mode of action of compound 1a, was identified by mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs) analysis using thin layer chromatography. Dose dependent experiments were conducted by over-expressing components of FAS-II in M. bovis BCG to confirm the target. Ligand binding using intrinsic tryptophan assay and molecular docking were used to further validate the target.

Results

MAMEs and FAMEs analysis showed dose-dependent reduction of MAMEs with the overall abundance of FAMEs suggesting that compound 1a targets mycolic acid biosynthesis. Direct binding of 1a to InhA was observed using an intrinsic tryptophan fluorescence binding assay, and a 2-fold IC₅₀ shift was observed with an InhA overexpressing strain confirming InhA as the cellular target.

Conclusion

The chalcone 1a exhibits potent antimycobacterial activity, displays a good safety profile and is a direct inhibitor of InhA, a key component in mycolic acid synthesis, validating this series for further anti-TB drug development.

Mycobacterium tuberculosis, the bacterium responsible for tuberculosis, is the global leading cause of mortality from an infectious agent. Part of this success relies on the unique cell wall, which consists of a thick waxy coat with tightly packed layers of complexed sugars, lipids and peptides. This coat provides a protective hydrophobic barrier to antibiotics and the host’s defences, while enabling the bacterium to spread efficiently through sputum to infect and survive within the macrophages of new hosts. However, part of this success comes at a cost, with many of the current first- and second-line drugs targeting the enzymes involved in cell wall biosynthesis. The flip side of this coin is that resistance to these drugs develops either in the target enzymes or the activation pathways of the drugs, paving the way for new resistant clinical strains. This review provides a synopsis of the structure and synthesis of the cell wall and the major current drugs and targets, along with any mechanisms of resistance.

Proteins are secreted from eukaryotic cells by several mechanisms besides the well-characterized classical secretory system. Proteins destined to enter the classical secretory system contain a signal peptide for translocation into the endoplasmic reticulum. However, many proteins lacking a signal peptide are secreted nonetheless. Contrary to conventional belief, these proteins are not just released as a result of membrane damage leading to cell leakage, but are actively packaged for secretion in alternative pathways. They are called unconventionally secreted proteins, and the best-characterized are from fungi and mammals. These proteins have extracellular functions including cell signaling, immune modulation, as well as moonlighting activities different from their well-described intracellular functions. Among the pathways for unconventional secretion are direct transfer across the plasma membrane, release within plasma membrane-derived microvesicles, use of elements of autophagy, or secretion from endosomal/multivesicular body-related components. We review the fungal and metazoan unconventional secretory pathways and their regulation, and propose experimental criteria to identify their mode of secretion.

Mycobacterium chelonae is an environmental, non-tuberculous mycobacterial species, capable of causing infections in humans. Biofilm formation is a key strategy used by M. chelonae in colonising niches in the environment and in the host. We studied a water-air interface (pellicle) biofilm of M. chelonae using a wide array of approaches to outline the molecular structure and composition of the biofilm. Scanning electron micrographs showed that M. chelonae biofilms produced an extracellular matrix. Using a combination of biochemical analysis, Raman spectroscopy, and fluorescence microscopy, we showed the matrix to consist of proteins, carbohydrates, lipids and eDNA. Glucose was the predominant sugar present in the biofilm matrix, and its relative abundance decreased in late (established) biofilms. RNA-seq analysis of the biofilms showed upregulation of genes involved in redox metabolism. Additionally, genes involved in mycolic acid, other lipid and glyoxylate metabolism were also upregulated in the early biofilms.

Despite the importance of fungal cell walls as the principle determinant of fungal morphology and the defining element determining fungal interactions with other cells, few scalar models have been developed that reconcile chemical and microscopic attributes of its structure. The cell wall of the fungal pathogen Candida albicans is comprised of an amorphous inner skeletal layer of β(1,3)- and β(1,6)-glucan and chitin and an outer fibrillar layer thought to be dominated by highly mannosylated cell wall proteins. The architecture of these two layers can be resolved at the electron microscopy level, but the visualised structure of the wall has not yet been defined precisely in chemical terms. We have therefore examined the precise structure, location and molecular sizes of the cell wall components using transmission electron microscopy and tomography and tested predictions of the cell wall models using mutants and agents that perturb the normal cell wall structure. We demonstrate that the fibrils are comprised of a frond of N-linked outer chain mannans linked to a basal layer of GPI-proteins concentrated in the mid-wall region and that the non-elastic chitin microfibrils are cantilevered with sufficient lengths of non-fibrillar chitin and/or β-glucan to enable the chitin-glucan cage to flex, e.g. during morphogenesis and osmotic swelling. We present the first three-dimensional nano-scalar model of the C. albicans cell wall which can be used to test hypotheses relating to the structure–function relationships that underpin the pathobiology of this fungal pathogen.

The cell wall polymers wall teichoic acid (WTA) and lipoteichoic acid (LTA) are often modified with glycosyl and D-alanine residues. Recent studies have shown that a three-component glycosylation system is used for the modification of LTA in several Gram-positive bacteria including Bacillus subtilis and Listeria monocytogenes. In the L. monocytogenes 1/2a strain 10403S, the cytoplasmic glycosyltransferase GtlA is thought to use UDP-galactose to produce the C₅₅-P-galactose lipid intermediate, which is transported across the membrane by an unknown flippase. Next, the galactose residue is transferred onto the LTA backbone on the outside of the cell by the glycosyltransferase GtlB. Here we show that GtcA is necessary for the glycosylation of LTA in L. monocytogenes 10403S and B. subtilis 168 and we hypothesize that these proteins act as C₅₅-P-sugar flippases. With this we revealed that GtcA is involved in the glycosylation of both teichoic acid polymers in L. monocytogenes 10403S, namely WTA with N-acetylglucosamine and LTA with galactose residues. These findings indicate that the L. monocytogenes GtcA protein can act on different C₅₅-P-sugar intermediates. Further characterization of GtcA in L. monocytogenes led to the identification of residues essential for its overall function as well as residues, which predominately impact WTA or LTA glycosylation.