Evolutionary history of Mycobacterium leprae in the Pacific Islands

As one of the oldest known human diseases, leprosy or Hansen''s disease remains a public health concern around the world with over 200 000 new cases in 2018. Most human leprosy cases are caused by Mycobacterium leprae, but a small number of cases are now known to be caused by Mycobacterium lepromatosis, a sister taxon of M. leprae. The global pattern of genomic variation in M. leprae is not well defined. Particularly, in the Pacific Islands, the origins of leprosy are disputed. Historically, it has been argued that leprosy arrived on the islands during nineteenth century colonialism, but some oral traditions and palaeopathological evidence suggest an older introduction. To address this, as well as investigate patterns of pathogen exchange across the Pacific Islands, we extracted DNA from 39 formalin-fixed paraffin-embedded biopsy blocks dating to 1992–2016. Using whole-genome enrichment and next-generation sequencing, we produced nine M. leprae genomes dating to 1998–2015 and ranging from 4-63× depth of coverage. Phylogenetic analyses indicate that these strains belong to basal lineages within the M. leprae phylogeny, specifically falling in branches 0 and 5. The phylogeographical patterning and evolutionary dating analysis of these strains support a pre-modern introduction of M. leprae into the Pacific Islands.This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.     

Discovery of 5-substituent-N-arylbenzamide derivatives as potent,selective and orally bioavailable LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson’s disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.     

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.     

Abundant DNase I-Sensitive Bacterial DNA in Healthy Porcine Lungs and Its Implications for the Lung Microbiome

Human lungs are constantly exposed to bacteria in the environment, yet the prevailing dogma is that healthy lungs are sterile. DNA sequencing-based studies of pulmonary bacterial diversity challenge this notion. However, DNA-based microbial analysis currently fails to distinguish between DNA from live bacteria and that from bacteria that have been killed by lung immune mechanisms, potentially causing overestimation of bacterial abundance and diversity. We investigated whether bacterial DNA recovered from lungs represents live or dead bacteria in bronchoalveolar lavage (BAL) fluid and lung samples in young healthy pigs. Live bacterial DNA was DNase I resistant and became DNase I sensitive upon human antimicrobial-mediated killing in vitro. We determined live and total bacterial DNA loads in porcine BAL fluid and lung tissue by comparing DNase I-treated versus untreated samples. In contrast to the case for BAL fluid, we were unable to culture bacteria from most lung homogenates. Surprisingly, total bacterial DNA was abundant in both BAL fluid and lung homogenates. In BAL fluid, 63% was DNase I sensitive. In 6 out of 11 lung homogenates, all bacterial DNA was DNase I sensitive, suggesting a predominance of dead bacteria; in the remaining homogenates, 94% was DNase I sensitive, and bacterial diversity determined by 16S rRNA gene sequencing was similar in DNase I-treated and untreated samples. Healthy pig lungs are mostly sterile yet contain abundant DNase I-sensitive DNA from inhaled and aspirated bacteria killed by pulmonary host defense mechanisms. This approach and conceptual framework will improve analysis of the lung microbiome in disease.     

Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.     

Scavenging affects persistence of avian carcasses resulting from window collisions in an urban landscape

ABSTRACT Collisions with windows remain an important human‐related threat to bird survival in urban landscapes. Accurately estimating the magnitude of avian mortality at windows is difficult and may be influenced by many sources of error, such as scavenging of carcasses. Failure to account for removal of carcasses by scavengers can bias estimates of window mortality. We tested the hypothesis that carcass survival depends on local habitat factors known to influence scavenger behavior. Scavenger activity on bird carcasses was documented at 20 buildings in an urban landscape in northwestern Illinois for 1 week during each season of a year. Known‐fate models were used to relate carcass survival to local habitat composition and to evaluate temporal variation in survival. We also documented species of scavengers and the timing of scavenging using motion‐triggered cameras. Daily carcass survival was greater in winter than during spring, summer, and fall. Survival was related negatively to canopy cover (trees and shrubs within a 50‐m buffer) and window area, and positively to pavement cover. Using an exponential model of survival time, estimated mean time of survival of carcasses (t± SE) was 82.9 ± 11.7 d for winter and 11.8 ± 7.2 d for other seasons. Raccoons (Procyon lotor) scavenged more carcasses than other species. Our results suggest that (1) carcass survival times may be short at locations with preferred habitats of known scavengers and predictable sources of food, and (2) knowledge of scavenger distribution and activity can inform predictive models of persistence. In studies of bird‐window collisions, the influence of scavenger bias can be minimized by maintaining short time intervals between carcass searches. Search intervals can be inferred by estimating the number of days that a carcass should persist at a site, which can be calculated using predicted daily survival probabilities of carcasses at study buildings.     

The Bowman-Birk inhibitor reactive site loop sequence represents an independent structural beta-hairpin motif

We have determined the NMR structure in aqueous solution of a disulphide-cyclised 11-residue peptide that forms a stable beta-hairpin, incorporating a type VIb beta-turn. The structure is found to be extremely well ordered for a short peptide, with the 30 lowest energy simulated annealing structures having an average pairwise r.m.s. deviation of only 0.36 A over the backbone. All but three side-chains adopt distinct conformations, allowing a detailed analysis of their involvement in cross-strand interactions. The peptide sequence analysed originates from a previously reported study, which identified potent inhibitors of human leukocyte elastase from screening a combinatorial peptide library based on the short protein beta-sheet segment that forms the reactive site loop of Bowman-Birk inhibitors. A detailed comparison of the peptide's solution structure with the corresponding region in the whole protein structure reveals a very good correspondence not only for the backbone (r.m.s. deviation approximately 0.7 A) but also for the side-chains. This isolated beta-hairpin retains the biologically active "canonical conformation" typical of small serine proteinase inhibitor proteins, which explains why it retains inhibitory activity. Since the structural integrity is sequence-inherent and does not depend upon the presence of the remaining protein, this beta-hairpin represents an independent structural motif and so provides a useful model of this type of protein architecture and its relation to biological function. The relationship between the conformation of this beta-hairpin and its biological activity is discussed.     

The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis

One characteristic linking members of the synaptotagmin family to endocytosis is their ability to bind the heterotetrameric AP2 complex via their C2B domain. By using CD4/synaptotagmin 1 chimeras, we found that the internalization signal of synaptotagmin 1 lies at the extreme COOH-terminus of the protein and can function in the absence of the C2B domain that contains the AP2 binding site. However, although not essential for internalization, the C2B domain of synaptotagmin 1 appeared to control the recognition of the internalization motif. By mutagenesis, two sites have been identified that modify regulation by the C2B domain in the neuroendocrine PC12 cell line. Mutation of a dilysine motif in the beta sandwich core of the domain eliminates endocytosis. This site is known to be a site of protein-protein interaction. Mutations in the calcium binding region, or in its close proximity, also affect internalization in PC12 cells. In fibroblasts, the C2B domain inhibits the COOH-terminal internalization signal, resulting in an absence of internalization in those cells. Thus, internalization of synaptotagmin 1 is controlled by the presence of a latent internalization signal in the COOH-terminal region and a regulatory region in the C2B domain. We propose that internalization of synaptotagmin 1 is regulated in this way to allow it to couple the processes of endocytosis and calcium-mediated exocytosis in cells of the neuroendocrine lineage.     

β-Glutamate as a Substrate for Glutamine Synthetase

The conversion of β-glutamate to β-glutamine by archaeal and bacterial glutamine synthetase (GS) enzymes has been examined. The GS from Methanohalophilus portucalensis (which was partially purified) is capable of catalyzing the amidation of this substrate with a rate sevenfold less than the rate obtained with α-glutamate. Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for α-glutamate than β-glutamate as a substrate. All the archaeal enzymes were much less selective than the two bacterial GS (from Escherichia coli and Bacillus subtilis), whose specific activities towards β-glutamate were much smaller than rates with the α-isomer. These results are discussed in light of the observation that β-glutamate is accumulated as an osmolyte in many archaea while β-glutamine (produced by glutamine synthetase) is used as an osmolyte only in M. portucalensis.     

Specificity of transinhibition of amino acid transport in neurospora

Amino acid transport systems I and III in Neurospora are inhibited by amino acids in the intracellular pool (transinhibition). The transinhibition is system specific. The ability of an amino acid to transinhibit a transport system is highly correlated with its affinity for the system. The significance of the system specificity of transinhibition is discussed.