Requirement of sphingolipid alpha-hydroxylation for fungicidal action of syringomycin E

Syringomycin E is an antifungal cyclic lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. To understand the mechanism of action of syringomycin E, a novel resistant Saccharomyces cerevisiae strain, BW7, was isolated and characterized. Lipid analyses revealed that BW7 contained only the hydrophobic subspecies of sphingolipids that are normally minor components in wild type strains. This aberrant sphingolipid composition was the result of lack of alpha-hydroxylation of the amide-linked very long chain fatty acids, suggesting a defective sphingolipid alpha-hydroxylase encoded by the FAH1 gene. A yeast strain that lacks the FAH1 gene was resistant to syringomycin E, and failed to complement BW7. These results demonstrate that BW7 carries a mutation in the FAH1 gene, and that the lack of alpha-hydroxylated very long chain fatty acids in yeast sphingolipids confers resistance to syringomycin E.     

Pulmonary collectins in innate immunity of the lung

Pulmonary collectins, hydrophilic surfactant proteins A and D (SP-A and SP-D), have been implicated in the regulation of pulmonary host defence and inflammation. SP-A and SP-D directly interact with a variety of microorganisms including bacteria and viruses, and attenuate the growth of Gram-negative bacteria, Histoplasma capsulatum and Mycoplasma pneumoniae. The collectins are thought to contribute to bacterial clearance. These lectins augment the phagocytosis of the bacteria by macrophages. SP-A serves as an opsonin and stimulates the uptake of bacteria and bacillus Calmette-Guérin through a C1q receptor- and an SP-R210-mediated processes. The collectin also stimulates FcR- and CR1-mediated phagocytosis by activating the macrophages. In addition, SP-A and SP-D directly interact with macrophages and enhance the phagocytosis of Streptococcus pneumoniae and Mycobacterium by increasing cell surface localization of the phagocytic receptors, scavenger receptor A and mannose receptor. The collectins also modulate pulmonary inflammation. SP-A and SP-D bind to cell surface receptors including Toll-like receptors, SIRPalpha and calreticulin/CD91, and attenuate or enhance inflammation in a microbial ligand-specific manner. In this article we review the immunomodulatory functions of SP-A and SP-D and their possible mechanisms in direct actions on microbes, macrophage phagocytosis and modulation of inflammation.     

Pulmonary collectins and innate host defense of the lung

Surfactant proteins A and D (SP-A and SP-D) are members of the collectin family of polypeptides expressed in the respiratory tract that bind bacterial, fungal and viral pathogens, enhancing their opsonization and killing by phagocytic cells. Clearance of bacterial pathogens including group B streptococci, Haemophilus influenza, Pseudomonas aeruginosa and viral pathogens, respiratory syncytial virus, adenovirus and influenza A virus, was deficient in SP-A(-/-) mice. SP-A deficiency was associated with enhanced inflammation and synthesis of proinflammatory cytokines. SP-D(-/-) mice cleared these bacteria as efficiently as wild-type mice; however, clearance of viral pathogens was deficient in SP-D(-/-) mice and associated with increased inflammation. SP-A and SP-D play critical and distinct roles in the regulation of alveolar macrophage function and inflammation, contributing to innate defense of the lung.     

The carbohydrate recognition domain of collectins

Collectins are effector molecules of the innate immune system that play an important role in the first line of defence against bacteria, viruses and fungi. Most of their interactions with microorganisms are mediated through their carbohydrate recognition domain (CRD), which binds in a Ca(2+)-dependent manner to glycoconjugates. This domain is a well-known structure that is present in a larger group of proteins comprising the C-type lectin domain family. Collectins form a subgroup within this family based on the presence of a collagen domain and the trimerization of CRDs, which are essential for the ligand-binding properties of these proteins. The ligand specificity among the nine collectin members is significantly different as a result of both the structural organization of the trimers and specific sequence changes in the binding pocket of the CRD. In addition, some collectin members have additional features, such as N-linked glycosylation of CRD residues and additional loop structures within the CRD that have a large impact on their interaction with the glycoconjugates present on microorganisms or host cells. The availability of crystal structures of three members of the collectin family (surfactant proteins A and D and mannan-binding protein) provides an important tool for addressing the impact of these CRD differences on ligand binding. In this review, the structural differences and similarities between the CRDs of collectins are summarized and their relationship with their ligand-binding characteristics is discussed.     

Porcine pulmonary collectins show distinct interactions with influenza A viruses: role of the N-linked oligosaccharides in the carbohydrate recognition domain

Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals. Pigs can serve as important intermediate hosts for transmission of avian IAV strains to humans, and for the generation of reassortant strains; this may result in the appearance of new pandemic IAV strains in humans. We have studied the role of the porcine lung collectins surfactant proteins D and A (pSP-D and pSP-A), two important components of the innate immune response against IAV. Hemagglutination inhibition assays revealed that both pSP-D and pSP-A display substantially greater inhibitory activity against IAV strains isolated from human, swine, and horse, than lung collectins from other animal species. The more potent activity of pSP-D results from interactions mediated by the asparagine-linked oligosaccharide located in the carbohydrate recognition domain of pSP-D, which is absent in SP-Ds from other species characterized to date. Presence of this sialylated oligosaccharide moiety enhances the anti-influenza activity of pSP-D, as demonstrated by assays of viral aggregation, inhibition of infectivity, and neutrophil response to IAV. The greater hemagglutination inhibitory activity of pSP-A is due to porcine-specific structural features of the conserved asparagine-linked oligosaccharide in the carbohydrate recognition domain of SP-A. A more efficient lung collectin-mediated immune response against IAV in pigs may play a role in providing conditions by which pigs can act as "mixing vessel" hosts that can lead to the production of reassortant, pandemic strains of IAV.     

Studies on the mechanism of fungicidal action of crystal violet on mycelial felts of fusarium culmorum

Summary Crystal violet, when incorporated in culture media of mycelial felts of Fusarium culmorum, reduced, or in certain cases stopped, the uptake of nitrate-nitrogen, protein and peptide synthesis, and carbon dioxide output by the mycelial mats.Addition of cysteine hydrochloride or sodium glutamate to the culture media did not alleviate the toxicity of crystal violet.The possible mode of fungicidal action of crystal violet is discussed.     

Studies on the mechanism of fungicidal action of mercuric chloride on mycelial felts of Rhizoctonia solani

Summary Mercuric chloride induced strong inhibitory effect on the growth, respiration and carbohydrate synthesis by mycelial felts of R. solani. Such inhibitory effects can be antagonised by the amino acid cysteine when mixed with the toxin in the nutritive medium. Methionine failed to do so. The possible explanations for the inhibitory actions of mercuric chloride are thoroughly discussed.     

Studies in the mechanism of fungicidal action: V. Non-metallic and sodium dithiocarbamic acid derivatives

This investigation has as its object the elucidation of the mode of action of dithiocarbamic acid derivatives as fungicides, with die help of the theory of variability, heavy metal salts being dealt with separately.
The experiments were carried out using Macrospcrium sarcinaeforme as the test organism; the chemical materials used are listed.
Deviations from the experimental and mathematical techniques described in previous papers of this series are explained.
From the results it is inferred that: ( a ) dithiocarbamates are decomposed by the spores to a greater or lesser degree into amine and carbon disulphide, both being toxic; ( b ) the amines can be absorbed only in a combined form, which may be the dithiccarbamate itself but is more probably a derivative, possibly an ester; ( c ) the carbon disulphide probably acts through the mediation of a thiocarbonate; ( d ) thiuramsulphides act through being decomposed to dithiocarbamates (or their derivatives mentioned above). The monosulphides are largely, and the disulphides slightly, decomposed; in the latter case with reduction.
It is suggested that the greater toxicity of the methyl-derivatives over all others tested is due to the shortness of the hydrocarbon chain.
It is concluded that the role of the carbon disulphide is chiefly that of a chemical carrier of the amino group, but that, owing to the negative index of variation of the spores to the latter, the carbon disulphide must play an essential part in completing the toxicity.
It is suggested that heavy-metal dimemyldithiocarbamates may show promise against M. sarcinaeforme, but that others may be effective against other fungi.     

Molecular basis for fungicidal action of neothyonidioside, a triterpene glycoside from the sea cucumber, Australostichopus mollis

Neothyonidioside is a triterpene glycoside (TG) isolated from the sea cucumber, Australostichopus mollis, that is potently cytotoxic to S. cerevisiae, but does not permeabilize cellular membranes. We mutagenized S. cerevisiae and isolated a neothionidioside-resistant (neo(R)) strain. Using synthetic genetic array mapping and sequencing, we identified NCP1 as the resistance locus. Quantitative HPLC revealed that neo(R)/ncp1 mutants have reduced ergosterol content. Ergosterol added to growth media reversed toxicity, demonstrating that neothionidioside binds directly to ergosterol, similar to the polyene natamycin. Ergosterol synthesis inhibitors ketoconazole and atorvastatin conferred resistance to neothionidioside in a dose-dependent manner showing that a threshold ergosterol concentration is required for toxicity. A genome-wide screen of deletion mutants against neothionidioside revealed hypersensitivity of many of the component genes in the ESCRT complexes relating to multivesicular body formation. Confocal microscopy of cells stained with a vital dye showed blockage at this step. Thus, we propose neothionidioside may affect membrane curvature and fusion capability in the endosome-vacuole pathway.     

Novel fungicidal benzylsulfanyl-phenylguanidines

A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted benzylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungicidal concentration, MFC ? 10 μM for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl scaffold with an unsubstituted guanidine resulted in less active compounds (MFC = 50-100 μM), whereas substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Compounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms consisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication of the biofilm, BEC50 ? 121 μM for both biofilm setups). Compounds 665 and 666 combined potent fungicidal (MFC = 5 μM) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermidis ? 4 μM). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a mini-host model.     

Pulmonary collectins selectively permeabilize model bacterial membranes containing rough lipopolysaccharide

We have reported that Gram-negative organisms decorated with rough lipopolysaccharide (LPS) are particularly susceptible to the direct antimicrobial actions of the pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D). In this study, we examined the lipid and LPS components required for the permeabilizing effects of the collectins on model bacterial membranes. Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with or without rough Escherichia coli LPS (J5), smooth E. coli LPS (B5), or cholesterol, were loaded with self-quenching probes and exposed to native or oxidatively modified SP-A. Fluorescence that resulted from permeabilization of liposomes and diffusion of dyes was assessed by microscopy or fluorimetry. Human SP-A and melittin increased the permeability of J5 LPS/POPE liposomes, but not B5 LPS/POPE liposomes or control (POPE only) liposomes. At a human SP-A concentration of 100 microg/mL, the permeability of the J5 LPS/POPE membranes increased 4.4-fold (p < 0.02) compared to the control with no added SP-A. Rat SP-A and SP-D also permeabilized the J5-containing liposomes. Incorporation of cholesterol into J5 LPS/POPE liposomes at a POPE:cholesterol molar ratio of 1:0.15 blocked human SP-A or melittin-induced permeability (p < 0.05) compared to cholesterol-free liposomes. Exposure of human SP-A to surfactant lipid peroxidation blocked the permeabilizing activity of the protein. We conclude that SP-A permeabilizes phospholipid membranes in an LPS-dependent and rough LPS-specific manner, that the effect is neither SP-A- nor species-specific, and that oxidative damage to SP-A abolishes its membrane destabilizing properties. Incorporation of cholesterol into the membrane enhances resistance to permeabilization by SP-A, most likely by increasing the packing density and membrane rigidity.