Katherine J. Siddle

A genomic portrait of the genetic architecture and regulatory impact of microRNA expression in response to infection

MicroRNAs (miRNAs) are critical regulators of gene expression, and their role in a wide variety of biological processes, including host antimicrobial defense, is increasingly well described. Consistent with their diverse functional effects, miRNA expression is highly context dependent and shows marked changes upon cellular activation. However, the genetic control of miRNA expression in response to external stimuli and the impact of such perturbations on miRNA-mediated regulatory networks at the population level remain to be determined. Here we assessed changes in miRNA expression upon Mycobacterium tuberculosis infection and mapped expression quantitative trait loci (eQTL) in dendritic cells from a panel of healthy individuals. Genome-wide expression profiling revealed that ∼40% of miRNAs are differentially expressed upon infection. We find that the expression of 3% of miRNAs is controlled by proximate genetic factors, which are enriched in a promoter-specific histone modification associated with active transcription. Notably, we identify two infection-specific response eQTLs, for miR-326 and miR-1260, providing an initial assessment of the impact of genotype-environment interactions on miRNA molecular phenotypes. Furthermore, we show that infection coincides with a marked remodeling of the genome-wide relationships between miRNA and mRNA expression levels. This observation, supplemented by experimental data using the model of miR-29a, sheds light on the role of a set of miRNAs in cellular responses to infection. Collectively, this study increases our understanding of the genetic architecture of miRNA expression in response to infection, and highlights the wide-reaching impact of altering miRNA expression on the transcriptional landscape of a cell.

Bacterial Infection Drives the Expression Dynamics of microRNAs and Their isomiRs

The optimal coordination of the transcriptional response of host cells to infection is essential for establishing appropriate immunological outcomes. In this context, the role of microRNAs (miRNAs) – important epigenetic regulators of gene expression – in regulating mammalian immune systems is increasingly well recognised. However, the expression dynamics of miRNAs, and that of their isoforms, in response to infection remains largely unexplored. Here, we characterized the genome-wide miRNA transcriptional responses of human dendritic cells, over time, to various mycobacteria differing in their virulence as well as to other bacteria outside the genus Mycobacterium, using small RNA-sequencing. We detected the presence of a core temporal response to infection, shared across bacteria, comprising 49 miRNAs, highlighting a set of miRNAs that may play an essential role in the regulation of basic cellular responses to stress. Despite such broadly shared expression dynamics, we identified specific elements of variation in the miRNA response to infection across bacteria, including a virulence-dependent induction of the miR-132/212 family in response to mycobacterial infections. We also found that infection has a strong impact on both the relative abundance of the miRNA hairpin arms and the expression dynamics of miRNA isoforms. That we observed broadly consistent changes in relative arm expression and isomiR distribution across bacteria suggests that this additional, internal layer of variability in miRNA responses represents an additional source of subtle miRNA-mediated regulation upon infection. Collectively, this study increases our understanding of the dynamism and role of miRNAs in response to bacterial infection, revealing novel features of their internal variability and identifying candidate miRNAs that may contribute to differences in the pathogenicity of mycobacterial infections.

The epigenomic landscape of African rainforest hunter-gatherers and farmers

The genetic history of African populations is increasingly well documented, yet their patterns of epigenomic variation remain uncharacterized. Moreover, the relative impacts of DNA sequence variation and temporal changes in lifestyle and habitat on the human epigenome remain unknown. Here we generate genome-wide genotype and DNA methylation profiles for 362 rainforest hunter-gatherers and sedentary farmers. We find that the current habitat and historical lifestyle of a population have similarly critical impacts on the methylome, but the biological functions affected strongly differ. Specifically, methylation variation associated with recent changes in habitat mostly concerns immune and cellular functions, whereas that associated with historical lifestyle affects developmental processes. Furthermore, methylation variation—particularly that correlated with historical lifestyle—shows strong associations with nearby genetic variants that, moreover, are enriched in signals of natural selection. Our work provides new insight into the genetic and environmental factors affecting the epigenomic landscape of human populations over time.

The Red Queen's long race: human adaptation to pathogen pressure

Pathogens, and the infectious diseases they cause, have been paramount among the threats encountered by humans in their expansions throughout the globe. Numerous studies have identified immunity and host defence genes as being among the functions most strongly targeted by selection, most likely pathogen-driven. The dissection of the form and intensity of such selective pressures have increased our knowledge of the biological relevance of the underlying immunological mechanisms in host defence. Although the identities of the specific infectious agents imposing these selective pressures remain, in most cases, elusive, the impact of several pathogens, notably malaria and cholera, has been described. However, past selection against infectious diseases may have some fitness costs upon environmental changes, potentially leading to maladaptation and immunopathology.

Widespread Shortening of 3’ Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3’ UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.

Evidence of Ebola Virus Replication and High Concentration in Semen of a Patient During Recovery

In one patient over time, we found that concentration of Ebola virus RNA in semen during recovery is remarkably higher than blood at peak illness. Virus in semen is replication-competent with no change in viral genome over time. Presence of sense RNA suggests replication in cells present in semen.

Negative Selection by Spiral Inertial Microfluidics Improves Viral Recovery and Sequencing from Blood

In blood samples from patients with viral infection, it is often important to separate viral particles from human cells, for example, to minimize background in performing viral whole genome sequencing. Here, we present a microfluidic device that uses spiral inertial microfluidics with continuous circulation to separate host cells from viral particles and free nucleic acid. We demonstrate that this device effectively reduces white blood cells, red blood cells, and platelets from both whole blood and plasma samples with excellent recovery of viral nucleic acid. Furthermore, microfluidic separation leads to greater viral genome coverage and depth, highlighting an important application of this device in processing clinical samples for viral genome sequencing.

Widespread shortening of 3' untranslated regions and increased exon inclusion characterize the human macrophage response to infection

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with two live bacteria. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, and found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Finally, our results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results show that dynamic changes in RNA processing play a key role in the regulation of innate immune responses.

Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient’s clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.