The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.     

An introduction of a pyridine group into the structure of prolyl oligopeptidase inhibitors

A series of ionizable prolyl oligopeptidase inhibitors were developed through the introduction of a pyridyl group to the P3 position of the prolyl oligopeptidase inhibitor structure. The study was performed on previously developed prolyl oligopeptidase inhibitors with proline mimetics at the P2 position. The 3-pyridyl group resulted in equipotent compounds as compared to the parent compounds. It was shown that the pyridyl group improves water solubility and, in combination with a 5(R)-tert-butyl-l-prolyl group at the P2 position, good lipophilicity can be achieved.     

Global Distribution and Evolution of a Toxinogenic Burkholderia-Rhizopus Symbiosis

Toxinogenic endobacteria were isolated from a collection of Rhizopus spp. representing highly diverse geographic origins and ecological niches. All endosymbionts belonged to the Burkholderia rhizoxinica complex according to matrix-assisted laser desorption ionization-time of flight biotyping and multilocus sequence typing, suggesting a common ancestor. Comparison of host and symbiont phylogenies provides insights into possible cospeciation and horizontal-transmission events.Bacterial symbionts and their metabolic potential play essential roles for many organisms. They may benefit from improved fitness, survival, and even acquired virulence (7, 12, 22). In the course of our studies of the biosynthesis of rhizoxin, the causative agent of rice seedling blight (10), we found that the phytotoxin is produced not by the fungus Rhizopus microsporus but by symbiotic bacteria (Burkholderia rhizoxinica) that reside within the fungus cytosol (13, 15, 23). Furthermore, cloning and sequencing of the rhizoxin biosynthesis gene cluster revealed the molecular basis of bacterial toxin production (14). In sum, this represents an unparalleled example for a symbiosis in which a fungus harbors bacteria for the production of a virulence factor. In analogy, we found that the first reported “mycotoxins” from lower fungi, the highly toxic cyclopeptides rhizonin A and B (25, 28), are also produced by symbiotic bacteria (Burkholderia endofungorum) and not by the fungus (16). While both rhizoxins and rhizonins have been believed to promote zygomycoses (21), there is no indication for toxin-producing endosymbiotic bacteria in clinical isolates (18).In nature, toxin production plays a pivotal role in the development of the fungus-bacterium association. Studies of the evolution of host resistance indicate that the association resulted from a pathogenicity mutualism shift in insensitive zygomycetes (24). The fungus lost its ability to sporulate independently and became totally dependent on endobacteria for reproduction through spores, thus warranting the persistence of the symbiosis and its efficient distribution through vegetative spores (17).To gain a broader view of the occurrence, biosynthetic potential, and relationship of toxinogenic endofungal bacteria, we investigated a collection of Rhizopus spp. consisting of 20 isolates classified as R. microsporus (of which 13 belong to R. microsporus var. microsporus, four to R. microsporus var. chinensis, two to R. microsporus var. oligosporus, and one to R. microsporus var. rhizopodiformis), one isolate classified as Rhizopus sp., and one Rhizopus oryzae strain. We initially monitored the presence of bacterial symbionts by PCR using universal primers (16S rRNA genes) and rhizoxin production in all available Rhizopus strains. Liquid cultivation of fungi in production medium with and without antibiotic followed by organic solvent extraction yielded crude extracts that were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). In total, eight fungal strains were identified or confirmed as rhizoxin positive and thus expected to harbor endosymbionts. In all cases, this assumption was verified by PCR and confocal scanning microscopy. By means of an optimized protocol, we finally succeeded in the isolation and cultivation of all eight bacterial symbiont strains in pure cultures (isolates B1 to B8) (Table ​(Table11).

TABLE 1.

Fungal strains and their bacterial endosymbionts

Environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces

Background

Early microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. Recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. The extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. We addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development.

Results

Genetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. Analysis of over 3,000 16S rRNA sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in early-life environment. Pigs housed in a natural outdoor environment showed a dominance of Firmicutes, in particular Lactobacillus, whereas animals housed in a hygienic indoor environment had reduced Lactobacillus and higher numbers of potentially pathogenic phylotypes. Our analysis revealed a strong negative correlation between the abundance of Firmicutes and pathogenic bacterial populations in the gut. These differences were exaggerated in animals housed in experimental isolators. Affymetrix microarray technology and Real-time Polymerase Chain Reaction revealed significant gut-specific gene responses also related to early-life environment. Significantly, indoor-housed pigs displayed increased expression of Type 1 interferon genes, Major Histocompatibility Complex class I and several chemokines. Gene Ontology and pathway analysis further confirmed these results.

Conclusion

Early-life environment significantly affects both microbial composition of the adult gut and mucosal innate immune function. We observed that a microbiota dominated by lactobacilli may function to maintain mucosal immune homeostasis and limit pathogen colonization.     

How mathematical modelling elucidates signalling in Bacillus subtilis

Appropriate stimulus perception, signal processing and transduction ensure optimal adaptation of bacteria to environmental challenges. In the Gram‐positive model bacterium Bacillus subtilis signalling networks and molecular interactions therein are well‐studied, making this species a suitable candidate for the application of mathematical modelling. Here, we review systems biology approaches, focusing on chemotaxis, sporulation, σ^B‐dependent general stress response and competence. Processes like chemotaxis and Z‐ring assembly depend critically on the subcellular localization of proteins. Environmental response strategies, including sporulation and competence, are characterized by phenotypic heterogeneity in isogenic cultures. The examples of mathematical modelling also include investigations that have demonstrated how operon structure and signalling dynamics are intricately interwoven to establish optimal responses. Our review illustrates that these interdisciplinary approaches offer new insights into the response of B. subtilis to environmental challenges. These case studies reveal modelling as a tool to increase the understanding of complex systems, to help formulating hypotheses and to guide the design of more directed experiments that test predictions.     

Analysis of membrane interactions of antibiotic peptides using ITC and biosensor measurements

The interaction of the lantibiotic gallidermin and the glycopeptide antibiotic vancomycin with bacterial membranes was simulated using mass sensitive biosensors and isothermal titration calorimetry (ITC). Both peptides interfere with cell wall biosynthesis by targeting the cell wall precursor lipid II, but differ clearly in their antibiotic activity against individual bacterial strains. We determined the binding affinities of vancomycin and gallidermin to model membranes±lipid II in detail. Both peptides bind to DOPC/lipid II membranes with high affinity (K(D) 0.30 μM and 0.27 μM). Gallidermin displayed also strong affinity to pure DOPC membranes (0.53 μM) an effect that was supported by ITC measurements. A surface acoustic wave (SAW) sensor allowed measurements in the picomolar concentration range and revealed that gallidermin targets lipid II at an equimolar ratio and simultaneously inserts into the bilayer. These results indicate that gallidermin, in contrast to vancomycin, combines cell wall inhibition and interference with the bacterial membrane integrity for potent antimicrobial activity.     

Tracking of fast moving neuronal vesicles with ageladine A

O-Linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins is a ubiquitous post-translational modification in multicellular organisms studied so far. Since aberrant O-GlcNAcylation has a link with insulin resistance, it is important to establish the status of O-GlcNAcylation in differentiation of mesenchymal cells such as preadipocytes. In this study, we found a differentiation-dependent drastic increase in the level of O-GlcNAcylation in mouse 3T3-L1 preadipocytes. The occurrence of the increase in O-GlcNAcylation, which correlated with the expression of C/EBPα, was in part due to increased expression of O-GlcNAc transferase. In addition to the well-known O-GlcNAcylated proteins such as nucleoporins and vimentin, pyruvate carboxylase, long chain fatty acid-CoA ligase 1, and Ewing sarcoma protein were identified as the proteins which are heavily O-GlcNAcylated with the adipocyte differentiation. Both adipocyte differentiation and the differentiation-dependent increase in O-GlcNAcylation were blocked by 6-diazo-5-oxo-norleucine. These results suggest that O-GlcNAcylation particilates, at least in part, in adipogenesis.     

Alien hand syndrome: neural correlates of movements without conscious will

Background

The alien hand syndrome is a striking phenomenon characterized by purposeful and autonomous movements that are not voluntarily initiated. This study aimed to examine neural correlates of this rare neurological disorder in a patient with corticobasal degeneration and alien hand syndrome of the left hand.

Methodology/Principal Findings

We employed functional magnetic resonance imaging to investigate brain responses associated with unwanted movements in a case study. Results revealed that alien hand movements involved a network of brain activations including the primary motor cortex, premotor cortex, precuneus, and right inferior frontal gyrus. Conscious and voluntary movements of the alien hand elicited a similar network of brain responses but lacked an activation of the inferior frontal gyrus. The results demonstrate that alien and unwanted movements may engage similar brain networks than voluntary movements, but also imply different functional contributions of prefrontal areas. Since the inferior frontal gyrus was uniquely activated during alien movements, the results provide further support for a specific role of this brain region in inhibitory control over involuntary motor responses.

Conclusions/Significance

We discuss the outcome of this study as providing evidence for a distributed neural network associated with unwanted movements in alien hand syndrome, including brain regions known to be related to movement execution and planning as well as areas that have been linked to inhibition control (inferior frontal gyrus) and experience of agency (precuneus).     

Testosterone and Visceral Fat in Midlife Women: The Study of Women's Health Across the Nation (SWAN) Fat Patterning Study

Visceral fat (VF) increases with the menopause and is an independent predictor of the metabolic syndrome, diabetes, and cardiovascular disease (CVD) in women. Little is known about how hormonal changes during the menopausal transition are related to the increase in VF. We aimed to determine the relationship between bioavailable testosterone and VF in middle‐aged women at various stages of the menopausal transition and whether this relationship is independent of age and other CVD risk factors. The Study of Women's Health Across the Nation (SWAN) is a longitudinal, community‐based study. This report uses baseline data from a population‐based longitudinal ancillary study at the Chicago site to examine the cross‐sectional relationship between testosterone and computed tomography (CT)–assessed VF in women at different stages of the menopausal transition. Included are 359 women (47.2% black), aged 42–60 years, who were randomly selected from a complete community census in which a 72% participation rate was achieved. In multivariate models, bioavailable testosterone was associated with VF independent of age, race, percent total body fat, and other cardiovascular risk factors. Bioavailable testosterone was a stronger predictor than estradiol and was interchangeable in its strength of association with sex hormone–binding globulin (SHBG). As bioavailable testosterone was associated with VF even after adjusting for insulin resistance, this suggests that it plays an important role in regional fat distribution. Our findings may have direct implications in explaining the effect of menopause‐related testosterone predominance on VF accumulation and subsequent cardiovascular risk.