Single cell view of tumor microenvironment gradients in pleural mesothelioma.

Immunotherapies have shown great promise in pleural mesothelioma (PM), yet most patients still do not achieve significant clinical response, highlighting the importance of improving understanding of the tumor microenvironment (TME). Here, we utilized high-throughput, single-cell RNA-sequencing to de novo identify 54 expression programs and construct a comprehensive cellular catalogue of the PM TME. We found four cancer-intrinsic programs associated with poor disease outcome and a novel fetal-like, endothelial cell population that likely responds to VEGF signaling and promotes angiogenesis. Throughout cellular compartments, we observe substantial difference in the TME associated with a cancer-intrinsic sarcomatoid signature, including enrichment in fetal-like endothelial cells, CXCL9+ macrophages, cytotoxic, exhausted, and regulatory T cells, which we validated using imaging and bulk deconvolution analyses on independent cohorts. Finally, we show, both computationally and experimentally, that NKG2A-HLA-E interaction between NK and tumor cells represents an important new therapeutic axis in PM, especially for epithelioid cases.

Complete genome sequence of Vibrio parahaemolyticus ST36 strain MAVP-26, a clinical isolate from an oyster-borne human gastric infection.

A Pacific native lineage of Vibrio parahaemolyticus ST36 serotype O4:K12 was introduced into the Atlantic, which increased local source illnesses. To identify genetic determinants of virulence and ecological resiliency and track their transfer into endemic populations, we constructed a complete genome of a 2013 Atlantic-traced clinical isolate by hybrid assembly.

A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

A master regulator of opioid reward in the ventral prefrontal cortex.

In addition to their intrinsic rewarding properties, opioids can also evoke aversive reactions that protect against misuse. Cellular mechanisms that govern the interplay between opioid reward and aversion are poorly understood. We used whole-brain activity mapping in mice to show that neurons in the dorsal peduncular nucleus (DPn) are highly responsive to the opioid oxycodone. Connectomic profiling revealed that DPn neurons innervate the parabrachial nucleus (PBn). Spatial and single-nuclei transcriptomics resolved a population of PBn-projecting pyramidal neurons in the DPn that express µ-opioid receptors (µORs). Disrupting µOR signaling in the DPn switched oxycodone from rewarding to aversive and exacerbated the severity of opioid withdrawal. These findings identify the DPn as a key substrate for the abuse liability of opioids.

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

Long-Read DNA Sequencing: Recent Advances and Remaining Challenges.

DNA sequencing has revolutionized medicine over recent decades. However, analysis of large structural variation and repetitive DNA, a hallmark of human genomes, has been limited by short-read technology, with read lengths of 100-300 bp. Long-read sequencing (LRS) permits routine sequencing of human DNA fragments tens to hundreds of kilobase pairs in size, using both real-time sequencing by synthesis and nanopore-based direct electronic sequencing. LRS permits analysis of large structural variation and haplotypic phasing in human genomes and has enabled the discovery and characterization of rare pathogenic structural variants and repeat expansions. It has also recently enabled the assembly of a complete, gapless human genome that includes previously intractable regions, such as highly repetitive centromeres and homologous acrocentric short arms. With the addition of protocols for targeted enrichment, direct epigenetic DNA modification detection, and long-range chromatin profiling, LRS promises to launch a new era of understanding of genetic diversity and pathogenic mutations in human populations.

Bat pluripotent stem cells reveal unusual entanglement between host and viruses.

Bats are distinctive among mammals due to their ability to fly, use laryngeal echolocation, and tolerate viruses. However, there are currently no reliable cellular models for studying bat biology or their response to viral infections. Here, we created induced pluripotent stem cells (iPSCs) from two species of bats: the wild greater horseshoe bat (Rhinolophus ferrumequinum) and the greater mouse-eared bat (Myotis myotis). The iPSCs from both bat species showed similar characteristics and had a gene expression profile resembling that of cells attacked by viruses. They also had a high number of endogenous viral sequences, particularly retroviruses. These results suggest that bats have evolved mechanisms to tolerate a large load of viral sequences and may have a more intertwined relationship with viruses than previously thought. Further study of bat iPSCs and their differentiated progeny will provide insights into bat biology, virus host relationships, and the molecular basis of bats' special traits.

Hedgehog-interacting protein acts in the habenula to regulate nicotine intake.

Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the Hhip gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.

NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer.

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8+ T cells. In bladder tumors, NKG2A is acquired on CD8+ T cells later than PD-1 as well as other well-established immune checkpoints. NKG2A+ PD-1+ CD8+ T cells diverge from classically defined exhausted T cells through their ability to react to human leukocyte antigen (HLA) class I-deficient tumors using T cell receptor (TCR)-independent innate-like mechanisms. HLA-ABC expression by bladder tumors is progressively diminished as disease progresses, framing the importance of targeting TCR-independent anti-tumor functions. Notably, NKG2A+ CD8+ T cells are inhibited when HLA-E is expressed by tumors and partly restored upon NKG2A blockade in an HLA-E-dependent manner. Overall, our study provides a framework for subsequent clinical trials combining NKG2A blockade with other T cell-targeted immunotherapies, where tumors express higher levels of HLA-E.

Incident Cancer Risk and Signatures Among Older MUTYH Carriers: Analysis of Population-Based and Genomic Cohorts.

MUTYH carriers have an increased colorectal cancer risk in case-control studies, with loss of heterozygosity (LOH) as the presumed mechanism. We evaluated cancer risk among carriers in a prospective, population-based cohort of older adults. In addition, we assessed if cancers from carriers demonstrated mutational signatures (G:C>T:A transversions) associated with early LOH. We calculated incident risk of cancer and colorectal cancer among 13,131 sequenced study participants of the ASPirin in Reducing Events in the Elderly cohort, stratified by sex and adjusting for age, smoking, alcohol use, BMI, polyp history, history of cancer, and aspirin use. MUTYH carriers were identified among 13,033 participants in The Cancer Genome Atlas and International Cancer Genome Consortium, and somatic signatures of cancers were analyzed. Male MUTYH carriers demonstrated an increased risk for overall cancer incidence [multivariable HR, 1.66; 95% confidence interval (CI), 1.03-2.68; P = 0.038] driven by increased colorectal cancer incidence (multivariable HR, 3.55; 95% CI, 1.42-8.78; P = 0.007), as opposed to extracolonic cancer incidence (multivariable HR, 1.40; 95% CI, 0.81-2.44; P = 0.229). Female carriers did not demonstrate increased risk of cancer, colorectal cancer, or extracolonic cancers. Analysis of mutation signatures from cancers of MUTYH carriers revealed no significant contribution toward early mutagenesis from widespread G:C>T:A transversions among gastrointestinal epithelial cancers. Among cancers from carriers, somatic transversions associated with base-excision repair deficiency are uncommon, suggestive of diverse mechanisms of carcinogenesis in carriers compared with those who inherit biallelic MUTYH mutations. PREVENTION RELEVANCE: Despite absence of loss of heterozygosity in colorectal cancers, elderly male MUTYH carriers appeared to be at increased of colorectal cancer.

Dietary fructose improves intestinal cell survival and nutrient absorption.

Fructose consumption is linked to the rising incidence of obesity and cancer, which are two of the leading causes of morbidity and mortality globally1,2. Dietary fructose metabolism begins at the epithelium of the small intestine, where fructose is transported by glucose transporter type 5 (GLUT5; encoded by SLC2A5) and phosphorylated by ketohexokinase to form fructose 1-phosphate, which accumulates to high levels in the cell3,4. Although this pathway has been implicated in obesity and tumour promotion, the exact mechanism that drives these pathologies in the intestine remains unclear. Here we show that dietary fructose improves the survival of intestinal cells and increases intestinal villus length in several mouse models. The increase in villus length expands the surface area of the gut and increases nutrient absorption and adiposity in mice that are fed a high-fat diet. In hypoxic intestinal cells, fructose 1-phosphate inhibits the M2 isoform of pyruvate kinase to promote cell survival5-7. Genetic ablation of ketohexokinase or stimulation of pyruvate kinase prevents villus elongation and abolishes the nutrient absorption and tumour growth that are induced by feeding mice with high-fructose corn syrup. The ability of fructose to promote cell survival through an allosteric metabolite thus provides additional insights into the excess adiposity generated by a Western diet, and a compelling explanation for the promotion of tumour growth by high-fructose corn syrup.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Myeloid Cell-associated Resistance to PD-1/PD-L1 Blockade in Urothelial Cancer Revealed Through Bulk and Single-cell RNA Sequencing.

PURPOSE: To define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer. EXPERIMENTAL DESIGN: We pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures. RESULTS: We identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (Msc2IR) score. Single myeloid phagocytic cells with low Msc2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade-resistant metastatic urothelial cancer. CONCLUSIONS: The balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood.See related commentary by Drake, p. 4139.

A reference profile-free deconvolution method to infer cancer cell-intrinsic subtypes and tumor-type-specific stromal profiles.

BACKGROUND: Patient stratification based on molecular subtypes is an important strategy for cancer precision medicine. Deriving clinically informative cancer molecular subtypes from transcriptomic data generated on whole tumor tissue samples is a non-trivial task, especially given the various non-cancer cellular elements intertwined with cancer cells in the tumor microenvironment. METHODS: We developed a computational deconvolution method, DeClust, that stratifies patients into subtypes based on cancer cell-intrinsic signals identified by distinguishing cancer-type-specific signals from non-cancer signals in bulk tumor transcriptomic data. DeClust differs from most existing methods by directly incorporating molecular subtyping of solid tumors into the deconvolution process and outputting molecular subtype-specific tumor reference profiles for the cohort rather than individual tumor profiles. In addition, DeClust does not require reference expression profiles or signature matrices as inputs and estimates cancer-type-specific microenvironment signals from bulk tumor transcriptomic data. RESULTS: DeClust was evaluated on both simulated data and 13 solid tumor datasets from The Cancer Genome Atlas (TCGA). DeClust performed among the best, relative to existing methods, for estimation of cellular composition. Compared to molecular subtypes reported by TCGA or other similar approaches, the subtypes generated by DeClust had higher correlations with cancer-intrinsic genomic alterations (e.g., somatic mutations and copy number variations) and lower correlations with tumor purity. While DeClust-identified subtypes were not more significantly associated with survival in general, DeClust identified a poor prognosis subtype of clear cell renal cancer, papillary renal cancer, and lung adenocarcinoma, all of which were characterized by CDKN2A deletions. As a reference profile-free deconvolution method, the tumor-type-specific stromal profiles and cancer cell-intrinsic subtypes generated by DeClust were supported by single-cell RNA sequencing data. CONCLUSIONS: DeClust is a useful tool for cancer cell-intrinsic molecular subtyping of solid tumors. DeClust subtypes, together with the tumor-type-specific stromal profiles generated by this pan-cancer study, may lead to mechanistic and clinical insights across multiple tumor types.

Elucidation of de novo small insertion/deletion biology with parent-of-origin phasing.

The mechanisms underlying de novo insertion/deletion (indel) genesis, such as polymerase slippage, have been hypothesized but not well characterized in the human genome. We implemented two methodological improvements, which were leveraged to dissect indel mutagenesis. We assigned de novo variants to parent-of-origin (i.e., phasing) with low-coverage long-read whole-genome sequencing, achieving better phasing compared to short-read sequencing (medians of 84% and 23%, respectively). We then wrote an application programming interface to classify indels into three subtypes according to sequence context. Across three cohorts with different phasing methods (Ntrios = 540, all cohorts), we observed that one de novo indel subtype, change in copy count (CCC), was significantly correlated with father's (p = 7.1 × 10-4 ) but not mother's (p = .45) age at conception. We replicated this effect in three cohorts without de novo phasing (ppaternal = 1.9 × 10-9 , pmaternal = .61; Ntrios = 3,391, all cohorts). Although this is consistent with polymerase slippage during spermatogenesis, the percentage of variance explained by paternal age was low, and we did not observe an association with replication timing. These results suggest that spermatogenesis-specific events have a minor role in CCC indel mutagenesis, one not observed for other indel subtypes nor for maternal age in general. These results have implications for indel modeling in evolution and disease.

Reprioritization of biofilm metabolism is associated with nutrient adaptation and long-term survival of Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) is a human-restricted pathogen with an essential requirement for heme-iron acquisition. We previously demonstrated that microevolution of NTHI promotes stationary phase survival in response to transient heme-iron restriction. In this study, we examine the metabolic contributions to biofilm formation using this evolved NTHI strain, RM33. Quantitative analyses identified 29 proteins, 55 transcripts, and 31 metabolites that significantly changed within in vitro biofilms formed by RM33. The synthesis of all enzymes within the tryptophan and glycogen pathways was significantly increased in biofilms formed by RM33 compared with the parental strain. In addition, increases were observed in metabolite transport, adhesin production, and DNA metabolism. Furthermore, we observed pyruvate as a pivotal point in the metabolic pathways associated with changes in cAMP phosphodiesterase activity during biofilm formation. Taken together, changes in central metabolism combined with increased stores of nutrients may serve to counterbalance nutrient sequestration.

Identification of a DNA N6-Adenine Methyltransferase Complex and Its Impact on Chromatin Organization.

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.

Capture of complete ciliate chromosomes in single sequencing reads reveals widespread chromosome isoforms.

BACKGROUND: Whole-genome shotgun sequencing, which stitches together millions of short sequencing reads into a single genome, ushered in the era of modern genomics and led to a rapid expansion of the number of genome sequences available. Nevertheless, assembly of short reads remains difficult, resulting in fragmented genome sequences. Ultimately, only a sequencing technology capable of capturing complete chromosomes in a single run could resolve all ambiguities. Even "third generation" sequencing technologies produce reads far shorter than most eukaryotic chromosomes. However, the ciliate Oxytricha trifallax has a somatic genome with thousands of chromosomes averaging only 3.2 kbp, making it an ideal candidate for exploring the benefits of sequencing whole chromosomes without assembly. RESULTS: We used single-molecule real-time sequencing to capture thousands of complete chromosomes in single reads and to update the published Oxytricha trifallax JRB310 genome assembly. In this version, over 50% of the completed chromosomes with two telomeres derive from single reads. The improved assembly includes over 12,000 new chromosome isoforms, and demonstrates that somatic chromosomes derive from variable rearrangements between somatic segments encoded up to 191,000 base pairs away. However, while long reads reduce the need for assembly, a hybrid approach that supplements long-read sequencing with short reads for error correction produced the most complete and accurate assembly, overall. CONCLUSIONS: This assembly provides the first example of complete eukaryotic chromosomes captured by single sequencing reads and demonstrates that traditional approaches to genome assembly can mask considerable structural variation.

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples.

BACKGROUND: Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, 'ResPipe'. RESULTS: Taxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve < 1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment. CONCLUSIONS: Sequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available ( https://gitlab.com/hsgweon/ResPipe ).

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.