A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Abstract Background Recurrent acute otitis media (rAOM, recurrent ear infection) is a common childhood disease caused by bacteria termed otopathogens, for which current treatments have limited effectiveness. Generic probiotic therapies have shown promise, but seem to lack specificity. We hypothesised that healthy children with no history of AOM carry protective commensal bacteria that could be translated into a specific probiotic therapy to break the cycle of re-infection. We characterised the nasopharyngeal microbiome of these children (controls) in comparison to children with rAOM (cases) to identify potentially protective bacteria. As some children with rAOM do not appear to carry any of the known otopathogens, we also hypothesised that characterisation of the middle ear microbiome could identify novel otopathogens, which may also guide the development of more effective therapies. Results Middle ear fluids, middle ear rinses and ear canal swabs from the cases and nasopharyngeal swabs from both groups underwent 16S rRNA gene sequencing. The nasopharyngeal microbiomes of cases and controls were distinct. We observed a significantly higher abundance of Corynebacterium and Dolosigranulum in the nasopharynx of controls. Alloiococcus, Staphylococcus and Turicella were abundant in the middle ear and ear canal of cases, but were uncommon in the nasopharynx of both groups. Gemella and Neisseria were characteristic of the case nasopharynx, but were not prevalent in the middle ear. Conclusions Corynebacterium and Dolosigranulum are characteristic of a healthy nasopharyngeal microbiome. Alloiococcus, Staphylococcus and Turicella are possible novel otopathogens, though their rarity in the nasopharynx and prevalence in the ear canal means that their role as normal aural flora cannot be ruled out. Gemella and Neisseria are unlikely to be novel otopathogens as they do not appear to colonise the middle ear in children with rAOM

Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS1)

Summary: Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS1). We establish that the NTS1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination

Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314

Abstract The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor’s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape

Additional file 10: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S6. Procrustes analysis of MEF and MER samples. The dataset includes pairs of MEF and MER samples from the same ear of the same child. Samples with less than 1499 reads are excluded. The p-value is non-parametric and is based on 999 Monte Carlo simulations. (PNG 80 kb

Additional file 5: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S2. Procrustes analysis of raw and rarefied datasets. The rarefied dataset was subsampled at a threshold of 1499 reads per sample. The raw dataset excluded samples below this depth. P-values are non-parametric and are based on 999 Monte Carlo simulations. (PNG 174 kb

Additional file 2: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S1. Diagrammatic overview of the 16S rRNA gene data analysis pipeline. Names of the software or tools used are in red. The SILVA database replaced the default taxonomy database in QIIME (GreenGenes) as GreenGenes 13_8 version does not discriminate between Alloiococcus and Dolosigranulum. (PDF 366 kb

Additional file 9: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S5. Procrustes analysis of left and right ear samples. The dataset includes both MEF and MER samples in left/right ear pairs from the same child. Samples with less than 1499 reads are excluded. The p-value is non-parametric and is based on 999 Monte Carlo simulations. (PNG 91 kb

Additional file 3: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Table S1. Full taxonomy of all OTUs identified in this study. Taxonomy is from the SILVA database, v123 for QIIME. Taxonomy assigned to OTUs by UCLUST v1.2.22q within QIIME v1.9.1 (assign_taxonomy.py). (XLSX 13 kb

Additional file 11: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Table S4. All correlations between OTUs determined by SparCC. Correlations within each sample type are listed in separate sheets. This includes correlations between all possible pairs of OTUs found in the samples. P-values are non-parametric and were calculated as the proportion of times a correlation coefficient more extreme than the observed correlation coefficient occurred in 100 simulated datasets. (XLSX 443 kb

Additional file 1: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Questionnaire completed by families recruited to the study. (PDF 28 kb