Proteomic analysis of Aspergillus fumigatus – clinical implications

Introduction: Aspergillus fumigatus is a ubiquitous saprophytic fungus capable of producing small airborne spores, which are frequently inhaled by humans. In healthy individuals, the fungus is rapidly cleared by innate mechanisms, including immune cells. However, in individuals with impaired lung function or immunosuppression the spores can germinate and prompt severe allergic responses, and disease with limited or extensive invasiveness.

Areas covered: The traits that make A. fumigatus a successful colonizer and pathogen of humans are multi-factorial. Thus, a global investigative approach is required to elucidate the mechanisms utilized by the fungus to cause disease.

Expert commentary: In doing so, a better understanding of disease pathology can be achieved with improved therapeutic/diagnostic solutions, thereby improving patient outcome. Proteomic analysis permits such investigations and recent work has yielded insight into these mechanisms.     

Differential responses by two closely related native fishes to restoration actions

Globally, river degradation has decimated freshwater fish populations. To help reverse this trend in a southeastern Australia river, we used multiple restoration actions, including reintroduction of instream woody habitat, riparian revegetation, removal of a weir hindering fish movement, fencing out livestock, and controlling riparian weeds. We monitored the responses of native fish at the segment scale (20 km) and reach scale (0.3 km) over 7 years to assess the effectiveness of the different restoration strategies. Two closely related species, Murray cod Maccullochella peeli and trout cod Maccullochella macquariensis, increased at the restored segment compared with the control segment. However, inherent differences between river segments and low sample size hampered assessment of the mechanisms responsible for segment‐scale changes in fish abundance. In contrast, at the reach scale, only M. peeli abundance significantly increased in reaches supplemented with wood. These differential responses by 2 closely related fish species likely reflect species‐specific responses to increased habitat availability and enhanced longitudinal connectivity when the weir improved passage around a fishway. Changes in M. peeli abundance in segments supplemented with and without wood suggest an increase in carrying capacity and not simply a redistribution of individuals within the segment, facilitated the observed expansion. Our findings confirm the need to consider individual fish species' habitat preferences carefully when designing restoration interventions. Further, species‐specific responses to restoration actions provide waterway managers with precise strategies to target fish species for recovery and the potential to predict fish outcomes based on ecological preferences.     

The Impact of Seeding Method on Diversity and Plant Distribution in Two Restored Grasslands

Previous studies have compared grassland restoration techniques based on resulting species richness and composition. However, none have determined if different techniques generate different plant distributions in space, which may further impact restoration success. This study tests if there are quadrat‐scale (1 m²) differences between paired drilled and broadcast plantings in diversity, composition, and plant distributions. Higher competition intensity in and more contiguous spaces between rows in drill‐seeded restorations were hypothesized to result in larger patches of native grasses and exotic species. Two paired drill‐ and broadcast‐seeded plantings were sampled in June 2007 in Iowa, U.S.A. Within 10 quadrats in each planting, we measured species abundance with point intercept sampling and plant distributions by dividing the quadrat into 64 cells and recording the most abundant species in each cell. Drilled and broadcast plantings at both sites had similar Simpson’s diversity and evenness. However, the effect of planting type on species richness, composition, and plant distribution was site dependent. Native warm‐season grasses in one site, and exotic species in the second, occupied more space and were distributed in larger patches in drilled plantings. Furthermore, drilled canopies consistently captured more light than broadcast canopies. This suggests that initial differences in seed placement can affect resulting plant distributions, resource use, and potentially long‐term species turnover. Mechanisms structuring vegetation in these communities need to be further investigated to determine if this approach can provide more information on long‐term diversity maintenance in restorations than traditional measures.     

Membrane topology and sequence requirements for oil body targeting of oleosin

Oleosin protein is targeted to oil bodies via the endoplasmic reticulum (ER) and consists of a lipid-submerged hydrophobic (H) domain that is flanked by cytosolic hydrophilic domains. We investigated the relationship between oleosin ER topology and its subsequent ability to target to oil bodies. Oleosin variants were created to yield differing ER membrane topologies and tagged with a reporter enzyme. Localisation was assessed by fractionation after transient expression in embryonic cells. Membrane-straddled topologies with N-terminal sequence in the ER lumen and C-terminal sequence in the cytosol were unable to target to oil bodies efficiently. Similarly, a translocated topology with only ER membrane and lumenal sequence was unable to target to oil bodies efficiently. Both topology variants accumulated proportionately higher in ER microsomal fractions, demonstrating a block in transferring from ER to oil bodies. The residual oil body accumulation for the inverted topology was shown to be because of partial adoption of native ER membrane topology, using a reporter variant, which becomes inactivated by ER-mediated glycosylation. In addition, the importance of H domain sequence for oil body targeting was assessed using variants that maintain native ER topology. The central proline knot motif (PKM) has previously been shown to be critical for oil body targeting, but here the arms of the H domain flanking this motif were shown to be interchangeable with only a moderate reduction in oil body targeting. We conclude that oil body targeting of oleosin depends on a specific ER membrane topology but does not require a specific sequence in the H domain flanking arms.     

Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe

O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.     

O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase

O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.     

Antimicrobial resistance profiling and DNA Amplification Fingerprinting (DAF) of thermophilic Campylobacter spp. in human, poultry and porcine samples from the Cork region of Ireland

Antimicrobial resistance (R) typing and DNA Amplification Fingerprinting (DAF) of a random collection of 84 Irish thermophilic Campylobacter isolates is described. The collection included human, veterinary (porcine) and poultry isolates cultured between 1996 and 1998 in the Cork region of Ireland. Biochemical and molecular methods were used to identify Campylobacter jejuni and Camp. coli. Many of these isolates were simultaneously resistant to several common antimicrobial agents. In particular, resistance to ampicillin, spectinomycin, sulphafurazole and tetracycline was common. A total of 74 DAF profiles was identified among the study collection, showing a high degree of diversity. Dendrogram analysis of the DNA patterns identified three main clusters at the 50% similarity level, which included two clusters of Camp. coli and a third containing a mixture of Camp. jejuni and Camp. coli.     

Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules

Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose and O-linked glucose saccharides, two unusual forms of O-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha2,3-Gal-beta1, 4-GlcNAc-beta1,3-Fuc-alpha1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represents the second protein discovered with an elongated form of O-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.