Fructose transport-deficient Staphylococcus aureus reveals important role of epithelial glucose transporters in limiting sugar-driven bacterial growth in airway surface liquid.

Hyperglycaemia as a result of diabetes mellitus or acute illness is associated with increased susceptibility to respiratory infection with Staphylococcus aureus. Hyperglycaemia increases the concentration of glucose in airway surface liquid (ASL) and promotes the growth of S. aureus in vitro and in vivo. Whether elevation of other sugars in the blood, such as fructose, also results in increased concentrations in ASL is unknown and whether sugars in ASL are directly utilised by S. aureus for growth has not been investigated. We obtained mutant S. aureus JE2 strains with transposon disrupted sugar transport genes. NE768(fruA) exhibited restricted growth in 10 mM fructose. In H441 airway epithelial-bacterial co-culture, elevation of basolateral sugar concentration (5-20 mM) increased the apical growth of JE2. However, sugar-induced growth of NE768(fruA) was significantly less when basolateral fructose rather than glucose was elevated. This is the first experimental evidence to show that S. aureus directly utilises sugars present in the ASL for growth. Interestingly, JE2 growth was promoted less by glucose than fructose. Net transepithelial flux of D-glucose was lower than D-fructose. However, uptake of D-glucose was higher than D-fructose across both apical and basolateral membranes consistent with the presence of GLUT1/10 in the airway epithelium. Therefore, we propose that the preferential uptake of glucose (compared to fructose) limits its accumulation in ASL. Pre-treatment with metformin increased transepithelial resistance and reduced the sugar-dependent growth of S. aureus. Thus, epithelial paracellular permeability and glucose transport mechanisms are vital to maintain low glucose concentration in ASL and limit bacterial nutrient sources as a defence against infection

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

<p>Abstract</p> <p>Background</p> <p>Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail.</p> <p>Findings</p> <p>To address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment.</p> <p>Conclusions</p> <p>When considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.</p

Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients

Analysis of ecological thresholds in a temperate forest undergoing dieback.

Positive feedbacks in drivers of degradation can cause threshold responses in natural ecosystems. Though threshold responses have received much attention in studies of aquatic ecosystems, they have been neglected in terrestrial systems, such as forests, where the long time-scales required for monitoring have impeded research. In this study we explored the role of positive feedbacks in a temperate forest that has been monitored for 50 years and is undergoing dieback, largely as a result of death of the canopy dominant species (Fagus sylvatica, beech). Statistical analyses showed strong non-linear losses in basal area for some plots, while others showed relatively gradual change. Beech seedling density was positively related to canopy openness, but a similar relationship was not observed for saplings, suggesting a feedback whereby mortality in areas with high canopy openness was elevated. We combined this observation with empirical data on size- and growth-mediated mortality of trees to produce an individual-based model of forest dynamics. We used this model to simulate changes in the structure of the forest over 100 years under scenarios with different juvenile and mature mortality probabilities, as well as a positive feedback between seedling and mature tree mortality. This model produced declines in forest basal area when critical juvenile and mature mortality probabilities were exceeded. Feedbacks in juvenile mortality caused a greater reduction in basal area relative to scenarios with no feedback. Non-linear, concave declines of basal area occurred only when mature tree mortality was 3-5 times higher than rates observed in the field. Our results indicate that the longevity of trees may help to buffer forests against environmental change and that the maintenance of old, large trees may aid the resilience of forest stands. In addition, our work suggests that dieback of forests may be avoidable providing pressures on mature and juvenile trees do not pass critical thresholds

A Novel Protein Isoform of the Multicopy Human NAIP Gene Derives from Intragenic Alu SINE Promoters

The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod-Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase-sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene

Identification of the CRE-1 Cellulolytic Regulon in Neurospora crassa

Background: In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and b-galactosidase. Methodology/Principal Findings: Here we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Dcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Dcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 59-SYGGRG-39 motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Dcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion

Impacts of soil conditions and light availability on natural regeneration of Norway spruce Picea abies (L.) H. Karst. in low-elevation mountain forests

& Key message Natural regeneration of P. abies (L.) H. Karst. may reach high densities in lower mountain elevations. The highest densities were found in sites with moderate light availability, with low pH, and not near the riverbank. However, age-height classes differed in the predicted magnitude of response, but were consistent in response directions. Mosses and understory species typical of coniferous forests were positively correlated with regeneration density. & Context Norway spruce Picea abies (L.) H. Karst. in Central Europe is at risk under climate change scenarios, particularly in mountain regions. Little is known about the impact of environmental factors on the natural regeneration of P. abies in lowelevation mountain forests. & Aims We aimed to assess impacts of distance from the riverbank, soil pH, and light availability on natural P. abies regeneration. We hypothesized that (1) natural P. abiesregeneration would depend on light availability and soil pH and (2) there are understory plant species which may indicate the microsites suitable for natural regeneration of P. abies. & Methods The study was conducted in the Stołowe Mountains National Park (SW Poland, 600–800 m a.s.l.). We established 160 study plots (25 m2 ) for natural regeneration, light availability, soil pH, and understory vegetation assessment

Climate warming disrupts mast seeding and its fitness benefits in European beech

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. Many plants benefit from synchronous year-to-year variation in seed production, called masting. Masting benefits plants because it increases the efficiency of pollination and satiates predators, which reduces seed loss. Here, using a 39-year-long dataset, we show that climate warming over recent decades has increased seed production of European beech but decreased the year-to-year variability of seed production and the reproductive synchrony among individuals. Consequently, the benefit that the plants gained from masting has declined. While climate warming was associated with increased reproductive effort, we demonstrate that less effective pollination and greater losses of seeds to predators offset any benefits to the plants. This shows that an apparently simple benefit of climate warming unravels because of complex ecological interactions. Our results indicate that in masting systems, the main beneficiaries of climate-driven increases in seed production are seed predators, not plants