Coping with antibiotic resistant septicemic E. coli.
Eliora Z. Ron, Tel Aviv University, Isreal
Antibiotic resistant Escherichia coli are a major cause of mortality and morbidity and the main threat in hospital and community acquired infections. The emergence of septicemic, highly antibiotic resistant pandemic strains constitutes a strong warning signal that warrants the search for new ways to fight such bacteria. We use functional genomics to understand the pathogenesis of septicemic drug-resistant E. coli and search for potential avenues for combatting these infections.
Terabytes and mosquito bites: The genetics, genomics and molecular biology of olfaction in disease vector mosquitoes as a lens on the evolution of behavior.
Laurence J. Zwiebel, Vanderbilt University in Nashville, USA
Almost twenty percent of all infectious human diseases are vector-borne. Together, they are responsible for over 1 million deaths per annum. Prior to the advent of next generation sequencing (NGS), the genomic tool kit of medical entomologists was largely constrained by its reliance on inferential information gleaned from “academic model” insect species, most notably the fruit fly Drosophila melanogaster. Over the last several years, the decreasing costs of massively parallel sequencing technologies have facilitated the agnostic interrogation of insect vector genomes, giving medical entomologists access to an ever-expanding volume of high quality genomic and transcriptomic data. These resources have broadened the scope of functional and evolutionary studies and have provided new insights into the biology of human and veterinary disease vectors.
Genomic, RNAseq and functional data will be discussed concerning the identification, evolution and characterization of several large super-families of receptors that together make up essential elements of the peripheral chemosensory signal transduction cascades across vector and non-vector mosquitoes. These include conserved and highly divergent odorant receptors (ORs), variant ionotropic glutamate-like ionotropic receptors (IRs) and gustatory receptors (GRs) associated with olfactory signaling pathways in An. gambiae and other mosquitoes. Recent studies of the chemosensory apparatus in An. gambiae and other Anopheline mosquitoes relative to host-selection/location, oviposition preference and reproduction along with increasingly detailed functional studies of AgORs and AgIRs will be presented. These data have implications for understanding of phylogenetic relationships as well as the mechanistic elements of signaling and provide a role of peripheral signaling in this system. Understanding of the olfactory system of An. gambiae and other vector insects is informing efforts to develop novel approaches to modulate oviposition and human host (blood meal) preferences that are critical mosquito behaviors that drive vectorial capacity as well as next generation repellents and attractants for anti-malarial programs that target chemosensory receptors and the behavioral processes they control in vector mosquitoes.
This work has received generous support from the Bill and Melinda Gates Foundation, through the Grand Challenges Initiative, the US National Institute of Allergy and Infectious Disease (NIAID) and Vanderbilt University.
Selection imposed by pathogens and archaic introgression have shaped the human immune system
Lluis Quintana-Murci, Institut Pasteur, CNRS, France
Unravelling the contributions of host and environmental factors to inter-individual and inter-population variability in immune responses is crucial to understand immune pathology. There is growing biomedical interest in studies of the variation of the immune response and its determinants in the healthy population ¾ in a strategy known as systems or population immunology.
Here, I will present our recent studies aiming to dissect the genetic, non-genetic and evolutionary determinants of immune response variation. The role played by pathogens in shaping human immune diversity is clearly attested by population genetic studies, indicating that immunity and host defense functions are among those most frequently subject to natural selection, whether purifying, positive or balancing. Notably, we have shown that population adaptation to novel pathogen pressures can be facilitated by the acquisition, via admixture, of advantageous alleles from local “adapted” populations, such as Neanderthals or modern humans.
I will also discuss our work on how genetic variation, whether adaptive or not, affects the diversity of molecular phenotypes (i.e., gene expression-eQTL, alternative splicing-sQTL, and DNA methylation-meQTL), and highlight the importance of inferring the causality behind the detected associations between genetic, epigenetic and transcriptional variation. Finally, I will present our recent data on the respective contributions of genetic and non-genetic factors, such as age and sex, to the diversity of both transcriptional responses to microbial challenges and epigenetic marks.
This presentation will attempt to provide a glimpse into how population and functional genomic approaches can help to pinpoint evolutionarily important determinants of host immune responsiveness.
Understanding pathogenesis of infectious disease through analyses of host genetic risk factors
Jenefer M. Blackwell, University of Western Australia, Australia, University of Cambridge, UK
In 2007 genome-wide association studies (GWAS) were hailed as a major innovation in understanding the molecular mechanisms of complex diseases. We undertook a GWAS for visceral leishmaniasis caused by Leishmania donovani in India and L. chagasi in Brazil. Variation within the Class II region of HLA, specifically at positions encoding amino acids 11 and 13 of HLA-DRB1, achieved genome-wide significance that crossed the epidemiological divides of pathogen species and geography. We mapped epitopes and binding affinities across Leishmania vaccine candidates, and peptide epitopes captured from dendritic cells treated with Leishmania antigen. We observed greater peptide promiscuity in sequence motifs for 9-mer core epitopes predicted to bind to risk (*1404/*1301) compared to protective (*1501) DRB1 alleles. There was a higher frequency of basic AAs in DRB1*1404-/*1301-specific epitopes, compared to hydrophobic and polar AAs in DRB1*1501-specific epitopes, at anchor residues P4 and P6 which interact with residues at DRB1 position 11 and 13. Cured VL patients made robust interferon-g, tumour necrosis factor and interleukin-10 responses to captured epitopes, with DRB1*1501-captured epitopes resulting in interferon-g:IL10 ratios >2-fold compared to peptides based on DRB1*1301-captured epitopes. In another GWAS for rheumatic heart disease (RHD) following Group A Streptococcus (GAS) infection in Aboriginal Australians, the strongest genetic risk was associated with specific HLA-DQA1-DQB1 haplotypes. Human myosin cross-reactive N-terminal and B repeat epitopes of GAS M5/M6 surface proteins bind with higher affinity to DQA1/DQB1 alpha/beta dimers for risk than protective haplotypes, supporting molecular mimicry as the key mechanism of RHD pathogenesis. These studies demonstrate that results of GWAS provide statistical confidence in genetic associations and can lead to greater understanding of molecular mechanisms underpinning infectious disease pathogenesis.
Computational approaches to microbiome characterization and infectious disease dynamics
Keith A. Crandall, George Washington University, USA
Microbiome characterization has become an integral component to a wide variety of disease and treatment approaches. Through the collection of metagenomic sequence data from DNA and/or RNA samples isolated from host individuals, effective microbiome characterization can identify pathogens, link diversity to disease state, characterize treatment effects, and identify drug resistant variants. I present a computational platform for metagenomic sequence analysis to characterize microbiome diversity and test hypotheses about diversity associates with disease. I present results from both empirical studies and simulation studies characterizing the utility of our computational approach to characterizing microbiome diversity with metagenomic data and compare our approach to other leading packages. I demonstrate our computational tools with applications in endangered species conservation, agriculture, and a variety of aspects of human health. Finally, I present new bioinformatic approaches for analyzing metagenomic data for population genomic studies of infectious diseases. Specifically, I present a novel pipeline for conducting real-time phylodynamics analyses of HIV infection and present results from HIV dynamics, diversity, and drug resistance in Washington DC, USA.
One locus sequencing typing (OLST), anovel metagenomic approach to analyze intra-species abundance and diversity
Teresa M. Coque, Ramón y Cajal Institute for BioHealth Research (IRYCIS), Spain
OBJECTIVES. Abundance and diversity of human opportunistic pathogens is necessary to rank their risk for infection and for transmission of adaptive traits of Public Health interest (e.g. antimicrobial resistance, virulence). Sequencing based methods as multilocus sequence typing (MLST), and more recently, core genome sequence typing (cgMLST), have extensively been used to track high-risk clonal complexes of different species but they are not compatible with the current high-throughput sequencing technologies. Available metagenomic approaches (e.g. 16S RNA, shotgun metagenomics) lack sensitivity and specificity to detect minority populations and to distinguish intraspecies diversity, respectively. We propose an affordable metagenomic approach to accurately detect abundance and diversity of major human pathogens.
MATERIAL AND METHODS. OLST is an approach that combines the use of a novel bioinformatic software to find an accurate intra-species diversity descriptor, amplification of such markers by PCR and long-read sequencing using minION (Oxford Nanopore). The software identifies genes with a phylogenetic tree that resembles the core-genome evolution by analysing non-redundant genome databases using graph networks and clustering algorithms.
RESULTS. The method was developed and validated for Escherichia coli, one of the most studied and diverse human opportunistic. From the approximately 250 genes of the E. coli core genome, we selected 10 as good predictors of the species diversity, three of them validated by PCR and further sequencing. OLST has been tested for other species of relevance in human medicine including Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, salmonella enterica and Klebsiella pneumoniae.
CONCLUSIONS. OLST is a fast, affordable, and accurate targeted metagenomic approach which allows identifying and quantifying the intra-species diversity of microorganisms in metagenomic samples with a high depth and accuracy. This new method can be applied to any species and can be easily updated to growing genome databases. The larger the database, the better descriptor will be selected.
Molecular epidemiology and evolution in the human immunodeficiency virus
Fernando González Candelas, University of Valencia, Spain
Despite significant improvements, the AIDS epidemics is still a major problem for public health. The adoption of routine genetic analyses of resistance mutations prior to initiation of therapy has resulted in increasing numbers of partial genome sequences which can be used for additional for the molecular surveillance of the HIV epidemics at the local, regional, national or global levels.
One immediate application at the local/regional level is the characterization of transmission clusters and networks. We will show such an example with data from the Comunidad Valenciana (Spain) where a sample of > 1,800 sequences obtained in 2004-2014 was used to characterize several transmission networks, the largest of which included > 100 individuals from the same city (Valencia) and which had been unnoticed to local public health authorities.
Another challenging application is the identification of new putative recombination forms, following detailed phylogenetic analyses. We have used this approach, along with complete genome sequences, to identify rare circular recombinant forms (CRFs) and describe new unique recombinant forms (URFs), thus providing a better picture of the diversity in HIV-1 due to recombination. The use of phylogenetic incongruence tests to characterize recombinant forms results in a re-evaluation of currently described CRFs.
Complete genome sequences also allow a less biased inference of basic evolutionary parameters in the history of organisms, after removing the effect of specific genes/regions. We have used a large data set of complete HIV-1 genomes (subtypes A1, B, C, D, and G, and CRFs 01_AE and 02_AG) to evaluate the evolutionary rate and time to the most recent common ancestor (tMRCA) for each gene and subtype or CRFs combination. The differences among these combinations can be explained by the action of natural selection and the effect of epidemic dynamics on these genomes.
Advances in reconstructing pathogen trait evolutionary history: New approaches and applications.
Philippe Lemey, University of Leuven, Belgium
The field of phylodynamics has witnessed a rich development of statistical inference tools with increasing levels of sophistication that can be applied to address a variety of questions about the evolution and epidemiology of viruses. The central premise of the field is that viruses generally evolve so rapidly that epidemic processes leave an imprint in their genomes. When focusing on deep evolutionary trees, a rich substitution history may confound time-measured analyses, whereas for short-term outbreaks, it may provide the necessary resolution for evolutionary reconstructions.
Here, I will illustrate these aspects on different viral examples. The Hepatitis B virus represents an example of a deep evolutionary history that has been difficult to date accurately using sequences sampled over the last decades. Recently, ancient DNA work has now resulted in the first HBV samples dating back thousands of years. Using molecular clock modelling that accommodates time-dependent evolutionary rates, I will show how recent rapid evolutionary rate estimates can be reconciled with the long-term evolutionary dynamics of the virus.
The 2013-2016 West African Ebola epidemic marked the start of real-time genomic sequencing. Using this example, I will illustrate that integrating various sources of information with genomic data promises to deliver more precise insights in infectious diseases. Finally, using recent work on Lassa virus in West Africa, I will further highlight how molecular epidemiology may impact on outbreak responses.
Massive human migration and spread of epidemic mycobacterium tuberculosis strains
Igor Mokrousov, St. Petersburg Pasteur Institute, St. Petersburg, Russia
Tuberculosis is ancient human disease that likely plagued humans since prehistory. Tuberculosis is remerging disease and most dangerous multidrug resistant Mycobacterium tuberculosis strains circulate in the high-incidence world regions. These strains are justly regarded as global health concern due to putative epidemic dissemination worldwide. However, the real situation is more complex and nuanced.
In my earlier work, I correlated evolutionary trajectories of the M. tuberculosis genotypes with distant events in human history. Here, I will analyze a recent impact of human migration on spread of epidemiologically successful M. tuberculosis clones that started to emerge in recent decades and even years and representing the most widespread East-Asian and Euro-American phylogenetic lineages. I compared their phylogeographic patterns with recent events in human history and migration. As a result, I propose three interconnected conclusions about the role of human migration and demographics in the spread of emerging and epidemic strains of M. tuberculosis. First, ordinary human exchange, such as travel is not enough to bring and settle down new M. tuberculosis strain in an autochthonous human population. In contrast, massive influx of migrants may change dramatically the population structure of the pathogen. Second, new emerging strain is emerging in its area of origin, where the parental strain was circulating. But not necessarily it will be successful and epidemic in genetically/ethnically different population. Third, to be efficiently imported to new location, a strain should be sufficiently highly prevalent in its country of origin. In summary, transmissibility of M. tuberculosis strains is conditional, but not absolute feature and is modulated by external conditions defined by human host.
Funding: Russian Science Foundation grant 14-14-00292.
Dynamics and evolution of dengue virus populations within their mosquito vector
Louis Lambrechts, Institut Pasteur, France; CNRS, France
Dengue viruses cause more human disease than any other arthropod-borne virus (arbovirus). Their main vector worldwide is the mosquito Aedes aegypti. I this talk, I will illustrate how studies of Ae. aegypti – dengue virus interactions at the within-host level can shed light on dengue epidemiological and evolutionary dynamics. First, I will show that infection dynamics within the mosquito vary among wild-type dengue virus isolates and that this genetic variation is predicted to affect the risk and magnitude of dengue outbreaks. Second, I will show that like other pathogens with high mutation rates and rapid replication, dengue viruses evolve during the infection of their vector. Dengue virus genetic diversity within the mosquito is shaped by genetic drift, purifying selection and vector genetic factors. Overall, accounting for within-host processes of vector-virus interactions provides important insights into arbovirus epidemiology and evolution.
Neisseria genomics: Current status and future perspectives.
Odile Harrison, University of Oxford, UK
The genus Neisseria is a group of Gram negative, oxidase positive aerobic b-Proteobacteria commonly associated with the dental and mucosal surfaces of animals and man (1, 2). Most of these species are harmless members of the commensal microbiota; however, the genus contains two important pathogens: Neisseria meningitidis, the meningococcus, and Neisseria gonorrhoeae, the gonococcus. The meningococcus is the causative agent of meningitis and septicaemia, although, this species is often referred to as an ‘‘accidental pathogen’’ in that the bacterium predominantly resides as a harmless commensal in the adult human nasopharynx, rarely becoming invasive. Disease results in an evolutionary dead end for the organism in that it does not lead to transmission (3). In contrast, the gonococcus is an obligate human pathogen with colonization resulting in a localized inflammatory response. More recently, there has been a worrying trend in the emergence of antimicrobial resistant gonococci worldwide with the prospect for this infection to be untreatable in the near future.
Genomes from both of these species have been increasingly sequenced allowing the population biology from these bacteria to be examined with unprecedented detail. This in turn provides opportunities for new prophylactic measures to be identified and/or new vaccine components to be developed. Whole genome sequences are relatively cheap and easy to obtain, however, complementary accessible, reproducible and rapid analysis techniques have been more challenging to reach. Access to such resources nevertheless is required if we are to maximise the potential benefit of WGS data in public health. Using the genus Neisseria as an exemplar, some of the bio-informatic approaches available to analyse WGS data will be presented, demonstrating how such data can be effectively visualised, understood and exploited.
1. Bennett JS, Bratcher HB, Brehony C, Harrison OB, Maiden MCJ. 2014. The Genus Neisseria, p. 881-900. In E. Rosenberg, E. F. DeLong, S. Lory, E. Stackebrandt, and F. Thompson (ed.), The Prokaryotes - Alphaproteobacteria and Betaproteobacteria, 4th ed. Springer Reference, Berlin Heidelberg.
2. Maiden MCJ and Harrison OB. 2016. Population and Functional Genomics of Neisseria Revealed with Gene-by-Gene Approaches. Journal of Clinical Microbiology 2016 Aug;54(8):1949-55
3. Caugant DA and Maiden MCJ. 2009. Meningococcal carriage and disease – population biology and evolution Vaccine, 27 (Suppl. 2), pp. B64-B70
Recent insights into the evolutionary history of arenaviruses
Manuela Sironi, Bioinformatic Unit of the Scientific Institute IRCCS E. MEDEA, Italy
Arenaviruses are enveloped negative-sense RNA viruses belonging to the Arenaviridae family. Several species of arenaviruses that infect mammals (Mammarenavirus genus) have been known for years. Some of these viruses cause severe hemorrhagic fevers in humans in Africa and Latin America. Divergent arenavirus lineages were described in snakes (Reptarenavirus genus) and fish (Antennavirus genus), much expanding the known diversity of arenaviruses. Recent efforts have also largely increased the availability of mammarenavirus genome sequences, allowing detailed molecular evolution analyses. Most mammarenaviruses establish acute or persistent infections in one of few rodent species and the majority of human mammarenavirus infections are caused by rodent-to-human transmission. Thus, the geographic range and evolutionary dynamics of mammarenaviruses are mainly determined by their natural hosts, although anthropogenic factors have likely played a role. Recent analyses indicated that the two major mammarenavirus lineages (Old World mammarenaviruses and New World mammarenaviruses) diverged less than ~45,000 years ago and that cospeciation did not contribute significantly to mammarenavirus association with their rodent hosts. During mammarenavirus speciation, the bulk of the viral genome evolved under purifying selection and structural protein features (e.g., solvent exposed surface area) were important determinants of site-wise evolutionary rate variation. However, the viral RNA polymerase (L protein) also represented a positive selection target during mammarenavirus and reptarenavirus speciation, as well as during the out-of-Nigeria migration of Lassa virus (LASV). This is interesting because changes in L affect viral persistence and virulence, at least in experimental settings. Despite these advances, several central questions remain unanswered. For instance the genetic determinants of virulence in different mammalian species are presently unknown and the reason(s) why mammarenavirus infection can determine very different disease severity among humans are obscure. Addressing these issues will be central to improve mammarenavirus infection treatment and prevention.
New insights into the origin and evolution of mycobacterium tuberculosis
Philip Supply, National Centre for Scientific Research, Institut Pasteur de Lille, France
M. tuberculosis (Mtb) remains the leading cause of death from a single infectious agent. New insights into the origin and factors driving this evolutionary success were gained through comparative and functional genomics of strain lineages of low and high epidemicity. We showed that exceptional tuberculosis (TB) clinical isolates from East Africa represent evolutionarily early branching lineages of the pathogen. These strains, called M. canettii, have larger genomes than Mtb, specific CRISPR-Cas systems, substantial diversity, and multiple traces of horizontal DNA exchange contrasting with the strong clonality of Mtb. The ability of these particular strains to exchange DNA was demonstrated, proving for the first time genetic transfers between TB bacilli. These differences are associated with a lower virulence/persistence in infection models, suggesting that Mtb emerged from an ancestral "M. canettii-like" pool by gain of pathogenicity mechanisms. One such mechanism consists of a change in cellular morphotype due to recombination in the pks5 locus in Mtb, revealing a key step in pathoadaptation. We also identified the epidemic expansion of 2 multidrug-resistant (MDR) Mtb clones over Eurasia, which began concurrently with the collapse of the public health system in the former USSR, largely explaining the association of Eurasian branches of the Beijing lineage of Mtb with MDR. In order to improve MDR detection, a next-generation sequencing-based in vitro test, directly applicable on clinical samples and called Deeplex®-MycTB, was developed. The assay uses deep sequencing of a single hi-plex amplicon mix for simultaneous species identification, genotyping and prediction of Mtb resistance to 13 drugs/drug classes. A secure web application, with integrated databases compiling drug resistance mutations characterized by WGS, allows fast and easy data analysis and reporting. This assay represents a new clinical tool for rapid individualization of TB treatments and expands the capacity for drug resistance surveillance in countries without culture facilities.
Supply P, et al. 2013. Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis. Nat Genet. 45:172–179.
Boritsch EC, et al. 2016. pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence. Nat Microbiol 1:15019.
Boritsch EC, et al. 2016. Key experimental evidence of chromosomal DNA transfer among selected tuberculosis-causing mycobacteria. Proc Natl Acad Sci U S A. 113:9876–9881.
Merker M, et al. 2015. Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage. Nat Genet. 47:242–9.
Walker TM, et al. 2015. Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study. Lancet Infect Dis. 15:1193–202.
The role of founder effects in speciation of parasitic protozoa: A unique Leishmania model in the Peruvian Andes.
Frederik Van den Broeck, Institute of Tropical Medicine Antwerp, Belgium
Introduction: Cutaneous leishmaniasis (CL) is the most common form of human leishmaniasis that produces skin lesions and leaves disfiguring scars, leading to social stigma and humiliation. Two causative agents of CL - Leishmania peruviana and L. braziliensis - present an extensive phenotypic diversity from an evolutionary, ecological and clinical point of view. The pathogenic L. braziliensis is a very old species that is endemic in the Amazonian lowlands East of the Peruvian Andes, extending throughout South- and Central-America. Its allopatric daughter species L. peruviana has a very restricted distribution and is only found in distinct eco-geographic zones on the western slopes of the Peruvian Andes, causing a disease of altitude that is characterized by negligible virulence. The advent of next-generation sequencing technologies offers an unprecedented resolution to study the contrasting evolutionary history of both Leishmania species in order to get insights into the genomic diversity underlying CL virulence.
Methods: We report whole-genome sequence data from 212 clinicalisolates from Peru, Bolivia and Brazil. Illumina sequence data were mapped against a novel PACBIO reference genome of L. braziliensis M2904, allowing us to study point and large-scale mutations.
Results: While L. braziliensis circulates in large panmictic populations that present extensive genomic diversity, we found that L. peruviana is divided into small isolated populations in separate pockets of suitable habitat. The origin of L. peruviana from a founder event of L. braziliensis parasites passing through the Andes is accompanied by a significant reduction in SNP diversity and large contractions/expansions of gene arrays that are implicated in parasite virulence.
Discussion: Our findings reveal major insights into the process of founder effect speciation and its impact on large-scale mutations that may underlie a loss in CL virulence.