Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment.

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

From Hype to Hope: The Gut Microbiota in Enteric Infectious Disease.

One of the clearest functions of the gut microbiota in humans is resistance to colonization by enteric bacterial pathogens. Reconstitution of the microbiota offers an exciting therapeutic approach, but great challenges must be overcome.

Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells.

Intestinal health relies on the immunosuppressive activity of CD4+ regulatory T (Treg) cells1. Expression of the transcription factor Foxp3 defines this lineage, and can be induced extrathymically by dietary or commensal-derived antigens in a process assisted by a Foxp3 enhancer known as conserved non-coding sequence 1 (CNS1)2-4. Products of microbial fermentation including butyrate facilitate the generation of peripherally induced Treg (pTreg) cells5-7, indicating that metabolites shape the composition of the colonic immune cell population. In addition to dietary components, bacteria modify host-derived molecules, generating a number of biologically active substances. This is epitomized by the bacterial transformation of bile acids, which creates a complex pool of steroids8 with a range of physiological functions9. Here we screened the major species of deconjugated bile acids for their ability to potentiate the differentiation of pTreg cells. We found that the secondary bile acid 3ß-hydroxydeoxycholic acid (isoDCA) increased Foxp3 induction by acting on dendritic cells (DCs) to diminish their immunostimulatory properties. Ablating one receptor, the farnesoid X receptor, in DCs enhanced the generation of Treg cells and imposed a transcriptional profile similar to that induced by isoDCA, suggesting an interaction between this bile acid and nuclear receptor. To investigate isoDCA in vivo, we took a synthetic biology approach and designed minimal microbial consortia containing engineered Bacteroides strains. IsoDCA-producing consortia increased the number of colonic RORγt-expressing Treg cells in a CNS1-dependent manner, suggesting enhanced extrathymic differentiation.

Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile.

The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.

Diversification and Evolution of Vancomycin-Resistant Enterococcus faecium during Intestinal Domination.

Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. This is particularly true in immunocompromised patients, where the damage to the microbiota caused by antibiotics can lead to VRE domination of the intestine, increasing a patient's risk for bloodstream infection. In previous studies we observed that the intestinal domination by VRE of patients hospitalized to receive allogeneic bone marrow transplantation can persist for weeks, but little is known about subspecies diversification and evolution during prolonged domination. Here we combined a longitudinal analysis of patient data and in vivo experiments to reveal previously unappreciated subspecies dynamics during VRE domination that appeared to be stable from 16S rRNA microbiota analyses. Whole-genome sequencing of isolates obtained from sequential stool samples provided by VRE-dominated patients revealed an unanticipated level of VRE population complexity that evolved over time. In experiments with ampicillin-treated mice colonized with a single CFU, VRE rapidly diversified and expanded into distinct lineages that competed for dominance. Mathematical modeling shows that in vivo evolution follows mostly a parabolic fitness landscape, where each new mutation provides diminishing returns and, in the setting of continuous ampicillin treatment, reveals a fitness advantage for mutations in penicillin-binding protein 5 (pbp5) that increase resistance to ampicillin. Our results reveal the rapid diversification of host-colonizing VRE populations, with implications for epidemiologic tracking of in-hospital VRE transmission and susceptibility to antibiotic treatment.

Dynamics of spore coat morphogenesis in Bacillus subtilis.

Spores of Bacillus subtilis are encased in a protective coat made up of at least 70 proteins. The structure of the spore coat has been examined using a variety of genetic, imaging and biochemical techniques; however, the majority of these studies have focused on mature spores. In this study we use a library of 41 spore coat proteins fused to the green fluorescent protein to examine spore coat morphogenesis over the time-course of sporulation. We found considerable diversity in the localization dynamics of coat proteins and were able to establish six classes based on localization kinetics. Localization dynamics correlate well with the known transcriptional regulators of coat gene expression. Previously, we described the existence of multiple layers in the mature spore coat. Here, we find that the spore coat initially assembles a scaffold that is organized into multiple layers on one pole of the spore. The coat then encases the spore in multiple co-ordinated waves. Encasement is driven, at least partially, by transcription of coat genes and deletion of sporulation transcription factors arrests encasement. We also identify the trans-compartment SpoIIIAH-SpoIIQ channel as necessary for encasement. This is the first demonstration of a forespore contribution to spore coat morphogenesis.

Physical interaction between coat morphogenetic proteins SpoVID and CotE is necessary for spore encasement in Bacillus subtilis.

Endospore formation by Bacillus subtilis is a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis.

Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.

Intestinal Bile Acids Induce a Morphotype Switch in Vancomycin-Resistant Enterococcus that Facilitates Intestinal Colonization.

Vancomycin-resistant Enterococcus (VRE) are highly antibiotic-resistant and readily transmissible pathogens that cause severe infections in hospitalized patients. We discovered that lithocholic acid (LCA), a secondary bile acid prevalent in the cecum and colon of mice and humans, impairs separation of growing VRE diplococci, causing the formation of long chains and increased biofilm formation. Divalent cations reversed this LCA-induced switch to chaining and biofilm formation. Experimental evolution in the presence of LCA yielded mutations in the essential two-component kinase yycG/walK and three-component response regulator liaR that locked VRE in diplococcal mode, impaired biofilm formation, and increased susceptibility to the antibiotic daptomycin. These mutant VRE strains were deficient in host colonization because of their inability to compete with intestinal microbiota. This morphotype switch presents a potential non-bactericidal therapeutic target that may help clear VRE from the intestines of dominated patients, as occurs frequently during hematopoietic stem cell transplantation.

Clostridium difficile colitis: pathogenesis and host defence.

Clostridium difficile is a major cause of intestinal infection and diarrhoea in individuals following antibiotic treatment. Recent studies have begun to elucidate the mechanisms that induce spore formation and germination and have determined the roles of C. difficile toxins in disease pathogenesis. Exciting progress has also been made in defining the role of the microbiome, specific commensal bacterial species and host immunity in defence against infection with C. difficile. This Review will summarize the recent discoveries and developments in our understanding of C. difficile infection and pathogenesis.

Complete Genome Sequence of Enterococcus faecium ATCC 700221.

We report the complete genome sequence of a vancomycin-resistant isolate of Enterococcus faecium derived from human feces. The genome comprises one chromosome of 2.9 Mb and three plasmids. The strain harbors a plasmid-borne vanA-type vancomycin resistance locus and is a member of multilocus sequencing type (MLST) cluster ST-17.

The Bacillus subtilis endospore: assembly and functions of the multilayered coat.

Sporulation in Bacillus subtilis involves an asymmetric cell division followed by differentiation into two cell types, the endospore and the mother cell. The endospore coat is a multilayered shell that protects the bacterial genome during stress conditions and is composed of dozens of proteins. Recently, fluorescence microscopy coupled with high-resolution image analysis has been applied to the dynamic process of coat assembly and has shown that the coat is organized into at least four distinct layers. In this Review, we provide a brief summary of B. subtilis sporulation, describe the function of the spore surface layers and discuss the recent progress that has improved our understanding of the structure of the endospore coat and the mechanisms of coat assembly.

A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat.

Bacillus subtilis spores are encased in a protein assembly called the spore coat that is made up of at least 70 different proteins. Conventional electron microscopy shows the coat to be organized into two distinct layers. Because the coat is about as wide as the theoretical limit of light microscopy, quantitatively measuring the localization of individual coat proteins within the coat is challenging. We used fusions of coat proteins to green fluorescent protein to map genetic dependencies for coat assembly and to define three independent subnetworks of coat proteins. To complement the genetic data, we measured coat protein localization at subpixel resolution and integrated these two data sets to produce a distance-weighted genetic interaction map. Using these data, we predict that the coat comprises at least four spatially distinct layers, including a previously uncharacterized glycoprotein outermost layer that we name the spore crust. We found that crust assembly depends on proteins we predicted to localize to the crust. The crust may be conserved in all Bacillus spores and may play critical functions in the environment.

Reduced growth of Drosophila neurofibromatosis 1 mutants reflects a non-cell-autonomous requirement for GTPase-Activating Protein activity in larval neurons.

Neurofibromatosis type 1 (NF1) is among the most common genetic disorders of humans and is caused by loss of neurofibromin, a large and highly conserved protein whose only known function is to serve as a GTPase-Activating Protein (GAP) for Ras. However, most Drosophila NF1 mutant phenotypes, including an overall growth deficiency, are not readily modified by manipulating Ras signaling strength, but are rescued by increasing signaling through the cAMP-dependent protein kinase A pathway. This has led to suggestions that NF1 has distinct Ras- and cAMP-related functions. Here we report that the Drosophila NF1 growth defect reflects a non-cell-autonomous requirement for NF1 in larval neurons that express the R-Ras ortholog Ras2, that NF1 is a GAP for Ras1 and Ras2, and that a functional NF1-GAP catalytic domain is both necessary and sufficient for rescue. Moreover, a Drosophila p120RasGAP ortholog, when expressed in the appropriate cells, can substitute for NF1 in growth regulation. Our results show that loss of NF1 can give rise to non-cell-autonomous developmental defects, implicate aberrant Ras-mediated signaling in larval neurons as the primary cause of the NF1 growth deficiency, and argue against the notion that neurofibromin has separable Ras- and cAMP-related functions.