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The two fungal human pathogens, Candida auris and Candida albicans, possess a variety of virulence mechanisms. Among them are the formation of biofilms to protect yeast against harsh conditions through the development of (pseudo)hyphae whilst also facilitating the invasion of host tissues. In recent years, increased rates of antifungal resistance have been associated with C. albicans and C. auris, posing a significant challenge for the effective treatment of fungal infections. In the course of our ongoing search for novel anti-infectives, six selected azaphilones were tested for their cytotoxicity and antimicrobial effects as well as for their inhibitory activity against biofilm and hyphal formation. This study revealed that rubiginosin C, derived from stromata of the ascomycete Hypoxylon rubiginosum, effectively inhibited the formation of biofilms, pseudohyphae, and hyphae in both C. auris and C. albicans without lethal effects. Crystal violet staining assays were utilized to assess the inhibition of biofilm formation, while complementary microscopic techniques, such as confocal laser scanning microscopy, scanning electron microscopy, and optical microscopy, were used to investigate the underlying mechanisms. Rubiginosin C is one of the few substances known to effectively target both biofilm formation and the yeast-to-hyphae transition of C. albicans and C. auris within a concentration range not affecting host cells, making it a promising candidate for therapeutic intervention in the future. [ABSTRACT FROM AUTHOR]

Interleukin-17 (IL-17)-secreting T cells of the T helper 17 ([T.sub.H]17) lineage play a pathogenic role in multiple inflammatory and autoimmune conditions and thus represent a highly attractive target for therapeutic intervention. We report that inhibition of acetyl-CoA carboxylase 1 (ACC1) restrains the formation of human and mouse [T.sub.H]17 cells and promotes the development of anti-inflammatory Foxp[3.sup.+] regulatory T ([T.sub.reg]) cells. We show that [T.sub.H]17 cells, but not [T.sub.reg] cells, depend on ACC1-mediated de novo fatty acid synthesis and the underlying glycolytic-lipogenic metabolic pathway for their development. Although [T.sub.H]17 cells use this pathway to produce phospholipids for cellular membranes, [T.sub.reg] cells readily take up exogenous fatty acids for this purpose. Notably, pharmacologic inhibition or T cell-specific deletion of ACC1 not only blocks de novo fatty acid synthesis but also interferes with the metabolic flux of glucose-derived carbon via glycolysis and the tricarboxylic acid cycle. In vivo, treatment with the ACC-specific inhibitor soraphen A or T cell-specific deletion of ACC1 in mice attenuates [T.sub.H]17 cell-mediated autoimmune disease. Our results indicate fundamental differences between [T.sub.H]17 cells and [T.sub.reg] cells regarding their dependency on ACC1-mediated de novo fatty acid synthesis, which might be exploited as a new strategy for metabolic immune modulation of [T.sub.H]17 cell-mediated inflammatory diseases., To develop from naive cells into distinct T cell lineages, activated T cells undergo a massive metabolic switch to cope with the demands of cell growth and multiple rounds of [...]

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/β-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy. Here the authors report that inhibition of de novo fatty acid synthesis by deleting the enzyme Acetyl-CoA-Carboxylase 1 in the intestinal epithelium results in the loss of crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells. [ABSTRACT FROM AUTHOR]

A study on the discrimination of the stable carbon isotopes by different microorganisms were made to determine whether ratios of carbon isotopes in microbial lipids can be used as indicators of substrate usage. Eleven strains of well known bacterial and fungal species with four different isotopically define carbon sources were grown and their isotope ratios of fatty acids of different lipid fractions were identified. Results show that the isotope ratio does not depend on the growth stage.

A study was conducted to isolate a novel fatty acid from the coryneform bacterial strain LMG 3820. A set of nuclear magnetic resonance data was utilized to determine the nature and substitution patterns of the glycerol moieties. Results indicated that different organisms supporting 16S rDNA sequences accommodate various fatty acid and lipid compositions. Findings also showed that the fatty acid's routine detection is influenced by the equivalent chain number.

High-pressure liquid chromatography was used to monitor N-cyclohexyldiazenium dioxide (HDO) concentration in isotope-labeled ([super 13]C) and nonlabeled ([super 12]C) Cu-HDO incorporated in wood sawdust mixed with soil. Cu-HDO as part of a wood preservative was observed to effectively decrease fungal wood decay and overall microbial respiration from soil.

The spatial and compositional structure and complex dynamics of the developing biofilm and its polychlorinated biphenyls (PCB)-degrading activity, leading to the identification of three stages of biofilm development is reported. The biofilm developing from PCB-contaminated soil on PCB droplets displayed a pronounced pattern of maturation not reported before from pure-culture experiments.

Fungi are present in the most diverse environments including the interior of plant tissues, living as endophytes without causing apparent damage. These endophytes are producers of secondary metabolites, also known as natural products, such as fungicides. Here, we evaluated the ethyl acetate fractions obtained from endophytic fungi isolated from plants in the genus Begonia. The fractions were submitted to inhibitory test against the plant pathogens Diaporthe phaseolorum and Colletotrichum gloeosporioides. From the 88 ethyl acetate fractions evaluated, 14.7 % inhibited C. gloeosporioides and 11.3 % inhibited D. phaseolorum. One fungal isolate displaying an active fraction was selected for chemical investigation. The fungus identified as Neopestalotiopsis sp., produced a compound that was active against D. phaseolorum, with a MIC of 312 µg mL-1 (1,695.3 µM). The compound was identified by mass spectrometry and ¹H NMR as the known compound fumiquinone B. The results highlight that the endophytes are capable of producing compounds that may be used to control plant pathogens. The compound fumiquinone B is reported for the first time as an antifungal agent against D. phaseolorum, a relevant plant pathogen worldwide. This is also the first report of the production of fumiquinone B by the genus Neopestalotiopsis. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is able to efficiently manipulate the host immune system establishing chronic infection, yet the underlying mechanisms of immune evasion are not fully understood. Evidence suggests that this pathogen interferes with host cell lipid metabolism to ensure its persistence. Fatty acid metabolism is regulated by acetyl-CoA carboxylase (ACC) 1 and 2; both isoforms catalyze the conversion of acetyl-CoA into malonyl-CoA, but have distinct roles. ACC1 is located in the cytosol, where it regulates de novo fatty acid synthesis (FAS), while ACC2 is associated with the outer mitochondrial membrane, regulating fatty acid oxidation (FAO). In macrophages, mycobacteria induce metabolic changes that lead to the cytosolic accumulation of lipids. This reprogramming impairs macrophage activation and contributes to chronic infection. In dendritic cells (DCs), FAS has been suggested to underlie optimal cytokine production and antigen presentation, but little is known about the metabolic changes occurring in DCs upon mycobacterial infection and how they affect the outcome of the immune response. We therefore determined the role of fatty acid metabolism in myeloid cells and T cells during Mycobacterium bovis BCG or Mtb infection, using novel genetic mouse models that allow cell-specific deletion of ACC1 and ACC2 in DCs, macrophages, or T cells. Our results demonstrate that de novo FAS is induced in DCs and macrophages upon M. bovis BCG infection. However, ACC1 expression in DCs and macrophages is not required to control mycobacteria. Similarly, absence of ACC2 did not influence the ability of DCs and macrophages to cope with infection. Furthermore, deletion of ACC1 in DCs or macrophages had no effect on systemic pro-inflammatory cytokine production or T cell priming, suggesting that FAS is dispensable for an intact innate response against mycobacteria. In contrast, mice with a deletion of ACC1 specifically in T cells fail to generate efficient T helper 1 responses and succumb early to Mtb infection. In summary, our results reveal ACC1-dependent FAS as a crucial mechanism in T cells, but not DCs or macrophages, to fight against mycobacterial infection. [ABSTRACT FROM AUTHOR]

Treating infections organized in biofilms is a challenge due to the resistance of the pathogens against antibiotics and host immune cells. Many fungi grow in a wet environment, favorable for the growth of bacterial biofilms, and we speculated that fungi possess some strategies to control these bacterial biofilms. A fungus identified as Hypoxylon fragiforme, was collected in the Harz Mountains, Germany, and its mycelial culture was fermented in different culture media for 67 days to test its biological potential against bacterial biofilms. Sclerin, sclerin diacid and its 3-methyl monoester (methyl 1-(5-hydroxy-6-carboxylic-2,3,4-trimethylphenyl) propionate) are here described for the first time from this fungus. Sclerin and its diacid interfered with the biofilm formation of the pathogen Staphylococcus aureus, inhibiting 86% and 80% of the biofilm at 256 μg mL-1, respectively, but not killing the bacterium. Interestingly, the monomethylester of sclerin diacid was inactive. Although these compounds did not possess any activity against a pre-formed biofilm, they prevented its formation at subtoxic concentrations. Furthermore, sclerin and its diacid displayed a high specificity against Staphylococcus aureus, indicating a good strategy against pathogenic biofilms when combined with antibiotics. [ABSTRACT FROM AUTHOR]

In the version of this article initially published online, the number 21.45, corresponding to the percentage of cells, was missing from the bottom right quadrant of the flow cytometry plot [...]

Pathogens embedded in biofilms are involved in many infections and are very difficult to treat with antibiotics because of higher resistance compared with planktonic cells. Therefore, new approaches for their control are urgently needed. One way to search for biofilm dispersing compounds is to look at defense strategies of organisms exposed to wet environments, which makes them prone to biofilm infections. It is reasonable to assume that mushrooms have developed mechanisms to control biofilms on their sporocarps (fruiting bodies). A preliminary screening for biofilms on sporocarps revealed several species with few or no bacteria on their sporocarps. From the edible mushroom Coprinus comatus where no bacteria on the sporocarp could be detected (3R,4S)-2-methylene-3, 4-dihydroxypentanoic acid 1,4-lactone, named coprinuslactone, was isolated. Coprinuslactone interfered with quorum-sensing and dispersed biofilms of Pseudomonas aeruginosa, where it also reduced the formation of the pathogenicity factors pyocyanin and rhamnolipid B. Coprinuslactone also damaged Staphylococcus aureus cells in biofilms at subtoxic concentrations. Furthermore, it inhibited UDP-Nacetylglucosamine enolpyruvyl transferase (MurA), essential for bacterial cell wall synthesis. These two modes of action ensure the inhibition of a broad spectrum of pathogens on the fruiting body but may also be useful for future clinical applications. [ABSTRACT FROM AUTHOR]

Most bacteria attach to surfaces where they form a biofilm, cells embedded in a complex matrix of polymers. Cells in biofilms are much better protected against noxious agents than free-living cells. As a consequence it is very difficult to control pathogens with antibiotics in biofilm infections and novel targets are urgently needed. One approach aims at the communication between cells to form and to maintain a biofilm, a process called quorum-sensing. Water soluble small-sized molecules mediate this process and a number of antagonists of these compounds have been found. In this review natural compounds and synthetic drugs which do not interfere with the classical quorum-sensing compounds are discussed. For some of these compounds the targets are still not known, but others interfere with the formation of exopolysaccharides, virulence factors, or cell wall synthesis or they start an internal program of biofilm dispersal. Some of their targets are more conserved among pathogens than the receptors for quorum sensing autoinducers mediating quorum-sensing, enabling a broader application of the drug. The broad spectrum of mechanisms, the diversity of bioactive compounds, their activity against several targets, and the conservation of some targets among bacterial pathogens are promising aspects for several clinical applications of this type of biofilm-controlling compound in the future. [ABSTRACT FROM AUTHOR]

Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5’-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado). [ABSTRACT FROM AUTHOR]

Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long-term intracellular lifestyle. Selected M ycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids ( PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and also with the specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments. [ABSTRACT FROM AUTHOR]

BACKGROUND: Asymptomatic bacterial colonisation of medical implants is increasingly recognised. The role of the often unusual bacteria is usually unknown however. OBJECTIVE AND METHODS: During a three-year period, patients presenting with clinically evident CIED lead endocarditis and/or pocket infections were included in a prospective database. Using 16s DNA analysis and sequencing, devices and pockets of consecutive patients with evident CIED infection were examined for the presence of bacterial species in addition to the ones grown in conventional culture in order to characterise their clinical role. RESULTS: During the study 77 of a total of 409 consecutive operations involving the explantation of a CIED were performed for clinically evident infection. 71 were included in the study and conventional extended culture and 16s DNA analysis performed and compared. In 42.3% of the patients bacteria were identified by DNA analysis in addition to the culture of the supposedly causative organisms. CONCLUSION: DNA analysis is more sensitive than conventional culture of swabs in detecting any, or indeed multiple bacterial strains in CIED infection. This may in future influence treatment strategies as e.g. the cohabitation of different strains seems common and is not reflected by classical culture results. Spread of infection along the leads was clearly demonstrated and this strongly supports the recommendation of complete removal of all CIED components in every form of CIED infection. The more unusual bacteria demonstrated here do not appear to play a significant clinical role as suspected earlier. [ABSTRACT FROM AUTHOR]

Poor sanitation, poor treatments of waste water, as well as catastrophic floods introduce pathogenic bacteria into rivers, infecting and killing many people. The goal of clean water for everyone has to be achieved with a still growing human population and their rapid concentration in large cities, often megacities. How long introduced pathogens survive in rivers and what their niches are remain poorly known but essential to control water-borne diseases in megacities. Biofilms are often niches for various pathogens because they possess high resistances against environmental stress. They also facilitate gene transfers of antibiotic resistance genes which become an increasing health problem. Beside biofilms, amoebae are carriers of pathogenic bacteria and niches for their survival. An overview about our current understanding of the fate and niches of pathogens in rivers, the multitude of microbial community interactions, and the impact of severe flooding, a prerequisite to control pathogens in polluted rivers, is given. [ABSTRACT FROM AUTHOR]

Abstract: Plant species exert strong effects on ecosystem functions and one of the emerging, and difficult to test hypotheses, is that plants alter soil functions through changing the community structure of soil microorganisms. We tested the hypothesis for atmospheric CH4 oxidation by using soil samples from a Siberian afforestation experiment and exposing them to 13C–CH4. We determined the activity of the soil methanotrophs under different tree species at three levels of initial CH4 concentration (30, 200 and 1000 ppm) thus distinguishing the activities of low- and high-affinity methanotrophs. Half of the samples were incubated with 13C-enriched CH4 (99.9%) and half with 12C–CH4. This allowed an estimation of the amount of 13C incorporated into individual PLFAs and determination of PLFAs of methanotrophs involved in CH4 oxidation at the different CH4 concentrations. Tree species strongly altered the activity of atmospheric CH4 oxidation without appearing to change the composition of high-affinity methanotrophs as evidenced by PLFA 13C labeling. The low diversity of atmospheric CH4 oxidizers, presumably belonging to the UCSα group, may explain the lack of tree species effects on the composition of soil methanotrophs. We submit that the observed tree species effects on atmospheric CH4 oxidation indicate an effect on biomass or cell-specific activities rather than by a community change and this may be related to the impact of the tree species on soil N cycling. [Copyright &y& Elsevier]

Marine pelagic redoxclines are zones of high dark CO2 fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO2 fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO2 incorporation in 13C-bicarbonate pulse experiments. The incorporation of 13C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in 13C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO2-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the 13C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO2-fixing Proteobacteria within this habitat. [ABSTRACT FROM AUTHOR]

Abstract: Highly virulent, slow-growing grey (SGG); moderately virulent, fast-growing salmon (FGS); and avirulent/weakly virulent, fast-growing grey (FGG) forms of Colletotrichum gloeosporioides have been described from yam (Dioscorea spp.), but little is known about their chemodiversity or the role of toxins in their pathogenesis. Secondary metabolite profiles in high performance tlc (hptlc) showed that the pathogenic SGG and FGS forms have a chemotype (A or B) that is distinct from the non-pathogenic FGG form (chemotype C). Crude extracts of 35-d-old Czapek–Dox yeast broth cultures of FGS and SGG isolates caused tissue necrosis on treated yam leaves but not those of FGG isolates. Extracts from uninoculated broth cultures showed no phytotoxic activity. Toxicity of the culture filtrate was not host specific and toxic substances were thermostable. Dioscorea genotypes with varying levels of resistance to anthracnose differed in their sensitivity to crude toxin extract of FGS (Cg33) and SGG (Cg25) isolates, indicating that these extracts may be useful in evaluating host resistance to anthracnose in vitro. Analysis of two toxin fractions unique to the pathogenic FGS and SGG forms using hlpc, mass spectrometry and nuclear magnetic resonance suggested the presence of a low molecular weight amide peptide. However, possibly due to low yield and the presence of impurities, the chemical structure of the compound(s) could not be fully elucidated. [Copyright &y& Elsevier]

Forest ecosystems assimilate more CO2 from the atmosphere and store more carbon in woody biomass than most nonforest ecosystems, indicating strong potential for afforestation to serve as a carbon management tool. However, converting grasslands to forests could affect ecosystem–atmosphere exchanges of other greenhouse gases, such as nitrous oxide and methane (CH4), effects that are rarely considered. Here, we show that afforestation on a well-aerated grassland in Siberia reduces soil CH4 uptake by a factor of 3 after 35 years of tree growth. The decline in CH4 oxidation was observed both in the field and in laboratory incubation studies under controlled environmental conditions, suggesting that not only physical but also biological factors are responsible for the observed effect. Using incubation experiments with 13CH4 and tracking 13C incorporation into bacterial phospholipid fatty acid (PLFA), we found that, at low CH4 concentrations, most of the 13C was incorporated into only two PLFAs, 18 : 1ω7 and 16 : 0. High CH4 concentration increased total 13C incorporation and the number of PLFA peaks that became labeled, suggesting that the microbial assemblage oxidizing CH4 shifts with ambient CH4 concentration. Forests and grasslands exhibited similar labeling profiles for the high-affinity methanotrophs, suggesting that largely the same general groups of methanotrophs were active in both ecosystems. Both PLFA concentration and labeling patterns indicate a threefold decline in the biomass of active methanotrophs due to afforestation, but little change in the methanotroph community. Because the grassland consumed CH4 at a rate five times higher than forest soils under laboratory conditions, we concluded that not only biomass but also cell-specific activity was higher in grassland than in afforested plots. While the decline in biomass of active methanotrophs can be explained by site preparation (plowing), inorganic N (especially NH4+) could be responsible for the change in cell-specific activity. Overall, the negative effect of afforestation of upland grassland on soil CH4 uptake can be largely explained by the reduction in biomass and to a lesser extent by reduced cell-specific activity of CH4-oxidizing bacteria. [ABSTRACT FROM AUTHOR]