Environmental and genetic influence on the rate and spectrum of spontaneous mutations in Escherichia coli.

Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterized the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a ΔluxS deletant strain - a gene with roles in both quorum sensing and the recycling of methylation products used in E. coli's DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive ΔluxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.

Drosophila versus Mycobacteria: A model for mycobacterial host-pathogen interactions.

Animal models have played an essential role in understanding the host-pathogen interactions of pathogenic mycobacteria, including the Mycobacterium tuberculosis and emerging nontuberculous mycobacteria (NTM) species such as M. avium and M. abscessus. Drosophila melanogaster has become a well-established model for the study of innate immunity and is increasingly being used as a tool to study host-pathogen interactions, in part due to its genetic tractability. The use of D. melanogaster has led to greater understanding of the role of the innate immune system in response to mycobacterial infection, including in vitro RNAi screens and in vivo studies. These studies have identified processes and host factors involved in mycobacterial infection, such as those required for cellular entry, those required to control or resist non-pathogenic mycobacteria, or factors that become dysregulated as a result of mycobacterial infection. Developments in genetic tools for manipulating mycobacterial genomes will allow for more detailed studies into how specific host and pathogen factors interact with one another by using D. melanogaster; however, the full potential of this model has not yet been reached. Here we provide an overview of how D. melanogaster has been used to study mycobacterial infection and discuss the current gaps in our understanding.

Effects of aging, cytomegalovirus infection, and EBV infection on human B cell repertoires.

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment.

The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.

Pathological features of West Nile and Usutu virus natural infections in wild and domestic animals and in humans: A comparative review.

Mosquito-borne flaviviruses are emerging pathogens with zoonotic potential. Due to the recent climate and environmental changes, they are spreading across Europe, becoming a major threat for public and veterinary health. West Nile virus (WNV) and Usutu virus (USUV) are arboviruses that are responsible for multiple disease outbreaks in different species of birds, reptiles, and mammals, including humans. This review reports and compares the clinical signs as well as the gross and microscopic pathological features during natural infection with WNV and USUV in wild and domestic animals, as well as in humans. The main objective of this comparative review is to delineate the common features and the specific differences that characterize WNV- and USUV-induced diseases in each group of species and to highlight the main gaps in knowledge that could provide insight for further investigation on the pathogenesis and neurovirulence of these viruses.

Novel approaches for the rapid development of rationally designed arbovirus vaccines.

Vector-borne diseases, including those transmitted by mosquitoes, account for more than 17% of infectious diseases worldwide. This number is expected to rise with an increased spread of vector mosquitoes and viruses due to climate change and man-made alterations to ecosystems. Among the most common, medically relevant mosquito-borne infections are those caused by arthropod-borne viruses (arboviruses), especially members of the genera Flavivirus and Alphavirus. Arbovirus infections can cause severe disease in humans, livestock and wildlife. Severe consequences from infections include congenital malformations as well as arthritogenic, haemorrhagic or neuroinvasive disease. Inactivated or live-attenuated vaccines (LAVs) are available for a small number of arboviruses; however there are no licensed vaccines for the majority of these infections. Here we discuss recent developments in pan-arbovirus LAV approaches, from site-directed attenuation strategies targeting conserved determinants of virulence to universal strategies that utilize genome-wide re-coding of viral genomes. In addition to these approaches, we discuss novel strategies targeting mosquito saliva proteins that play an important role in virus transmission and pathogenesis in vertebrate hosts. For rapid pre-clinical evaluations of novel arbovirus vaccine candidates, representative in vitro and in vivo experimental systems are required to assess the desired specific immune responses. Here we discuss promising models to study attenuation of neuroinvasion, neurovirulence and virus transmission, as well as antibody induction and potential for cross-reactivity. Investigating broadly applicable vaccination strategies to target the direct interface of the vertebrate host, the mosquito vector and the viral pathogen is a prime example of a One Health strategy to tackle human and animal diseases.

Individual variation in the germline Ig gene repertoire inferred from variable region gene rearrangements.

Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

A Journey to the Central Nervous System: Routes of Flaviviral Neuroinvasion in Human Disease.

Many arboviruses, including viruses of the Flavivirus genera, are known to cause severe neurological disease in humans, often with long-lasting, debilitating sequalae in surviving patients. These emerging pathogens impact millions of people worldwide, yet still relatively little is known about the exact mechanisms by which they gain access to the human central nervous system. This review focusses on potential haematogenous and transneural routes of neuroinvasion employed by flaviviruses and identifies numerous gaps in knowledge, especially regarding lesser-studied interfaces of possible invasion such as the blood-cerebrospinal fluid barrier, and novel routes such as the gut-brain axis. The complex balance of pro-inflammatory and antiviral immune responses to viral neuroinvasion and pathology is also discussed, especially in the context of the hypothesised Trojan horse mechanism of neuroinvasion. A greater understanding of the routes and mechanisms of arboviral neuroinvasion, and how they differ between viruses, will aid in predictive assessments of the neuroinvasive potential of new and emerging arboviruses, and may provide opportunity for attenuation, development of novel intervention strategies and rational vaccine design for highly neurovirulent arboviruses.

Mycobacterium abscessus pathogenesis identified by phenogenomic analyses.

The medical and scientific response to emerging and established pathogens is often severely hampered by ignorance of the genetic determinants of virulence, drug resistance and clinical outcomes that could be used to identify therapeutic drug targets and forecast patient trajectories. Taking the newly emergent multidrug-resistant bacteria Mycobacterium abscessus as an example, we show that combining high-dimensional phenotyping with whole-genome sequencing in a phenogenomic analysis can rapidly reveal actionable systems-level insights into bacterial pathobiology. Through phenotyping of 331 clinical isolates, we discovered three distinct clusters of isolates, each with different virulence traits and associated with a different clinical outcome. We combined genome-wide association studies with proteome-wide computational structural modelling to define likely causal variants, and employed direct coupling analysis to identify co-evolving, and therefore potentially epistatic, gene networks. We then used in vivo CRISPR-based silencing to validate our findings and discover clinically relevant M. abscessus virulence factors including a secretion system, thus illustrating how phenogenomics can reveal critical pathways within emerging pathogenic bacteria.

Cysteine-rich peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development.

Cell-cell communication in plants is essential for the correct co-ordination of reproduction, growth, and development. Studies to dissect this mode of communication have previously focussed primarily on the action of plant hormones as mediators of intercellular signalling. In animals, peptide signalling is a well-documented intercellular communication system, however, relatively little is known about this system in plants. In recent years, numerous reports have emerged about small, secreted peptides controlling different aspects of plant reproduction. Interestingly, most of these peptides are cysteine-rich, and there is convincing evidence suggesting multiple roles for related cysteine-rich peptides (CRPs) as signalling factors in developmental patterning as well as during plant pathogen responses and symbiosis. In this review, we discuss how CRPs are emerging as key signalling factors in regulating multiple aspects of vegetative growth and reproductive development in plants.

Central cell-derived peptides regulate early embryo patterning in flowering plants.

Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements.

B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual's secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.

Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing.

The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. In this work, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of barcoded amplicons were sequenced using long-read ultra deep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or re-emergence of such populations after treatment.

Identification of a rapid mammalian deadenylation-dependent decay pathway and its inhibition by a viral RNA element.

Cellular RNAs are subject to quality-control pathways that insure the fidelity of gene expression. We previously identified a 79 nt element, the ENE, that is essential for the nuclear accumulation of a viral polyadenylated nuclear (PAN) RNA. Here, we show that intron-less polyadenylated transcripts such as PAN RNA and beta-globin cRNA exhibit two-component exponential decay kinetics in which some transcripts are rapidly degraded (t(1/2) = approximately 15 min) while others decay more slowly (t(1/2) = approximately 3 hr). Inclusion of the ENE protects such transcripts from rapid decay in a poly(A)-dependent fashion. The ENE inhibits deadenylation and decay in nuclear extract and prevents deadenylation of naked RNA by a purified deadenylase, likely through snoRNA-like intramolecular hybridization with the poly(A) tail. The ENE causes increased accumulation of splicing-defective beta-globin pre-mRNAs in vivo. We propose that the ENE-controlled rapid-decay mechanism for polyadenylated transcripts comprises a nuclear pre-mRNA surveillance system in mammalian cells.