EVOLUTIONARY ANALYSES PROVIDE INSIGHT INTO HOST-PATHOGEN INTERACTIONS AND DIET-RELATED ADAPTATIONS

ABSTRACT INTRODUCTION Genetic diversity plays an important role in the survival and adaptability of all species. When a population environment (meaning, for instance, climatic conditions, pathogens, and food availability) changes, the population is subject to a selective pressure. Variation in the population gene pool provides variable traits which can be selected for, via natural selection, leading to an adaptive change to survive. Genetic diversity is generated by a combination of different evolutionary processes such as mutation, genetic drift, migration and natural selection. Natural selection leaves a distinctive molecular signature in genomes. Such molecular signatures can be detected with evolutionary tests that can be divided into those that search for selection at the inter-species level (e.g., human versus primates and mammals) and those that focus on within-species data (e.g., among human populations). In my work, I used evolutionary studies to analyze genes under different selective pressures. In a first study, I investigate the evolutionary history of genes possibly involved in diet adaptation. Changes in food availability and diet likely created strong selective pressures on multiple biological processes. In humans, the agricultural revolution favored carbohydrate consumption. I exploit the availability of genome sequences from different organisms, together with resequencing data of ancient DNA samples to perform a comprehensive evolutionary analysis of genes involved in sugar absorption/digestion at the brush-border and to test when adaptive alleles arose. In a second set of studies, I use evolutionary analyses to investigate the interaction between host proteins and viral/protozoan/bacterial protein products. Molecules that participate in immune response are expected to be engaged in a constant arms-race with pathogens and to harbour the molecular signatures of such a conflict. I thus investigate the evolutionary history of genes involved in immune defense, such as antiviral sensing proteins, genes with IFN-inducible properties and antiviral effectors. I also investigate the evolutionary history of the complement system, an innate immunity effector, and of bacterial-encoded complement- interacting proteins. Nevertheless, not only genes with specific defense function, but also molecules involved in central homeostatic processes may be engaged in genetic conflicts with pathogens. This is exemplified by (i) the sterol transporter NPC1, used as receptor for filoviruses entry and (ii) basigin, a multifunctional protein with a role in trophoblast function and in spermatogenesis which is used for erythrocyte invasion by Plasmodium falciparum. I therefore study the evolutionary history of these genes and of their viral/microbial interactors. AIM OF THE WORK The purpose of my project is to use evolutionary analyses to investigate and describe adaptive events at candidate genes subject to different selective pressures, in species ranging from mammals to viruses/protozoa/bacteria. In particular, I focus on inter-species and population genetics-phylogenetics analyses, with the aim to detect positive selection acting over long evolutionary timescales. MATERIALS AND METHODS Mammalian and pathogen coding gene sequences were retrieved from public databases (Ensembl, UCSC and NCBI) or obtained by direct sequencing. Information from the Neandertal and Denisova high-coverage genomes, as well as from a hunter-gatherer Mesolithic European from Spain and a Paleolithic Siberian was derived from previous works. Sequences were aligned using RevTrans 2.0 utility, PRANK, and unreliably aligned codons were then filtered using GUIDANCE. Since recombination can be mistaken as positive selection, all alignments were screened for the presence of recombination breakpoints using a specific software (GARD). To detect selection, codeml models were fitted to the data using different models of equilibrium codon frequencies. Sites under selection were identified using BEB and MEME. Branches and sites subject to episodic positive selection were identified using Bs-Rel or the branch-site tests from the codeml software. Evolutionary analysis in the human, chimpanzee and gorilla lineages was performed using a population genetics-phylogenetics approach. Analysis of 3D structures was used to infer the functional significance of positively selected sites. In order to assess the role of selected variants in HIV-1 susceptibility, and to evaluate gene expression in response to interferon alpha (IFN-α), genetic association analyses, in vitro HIV-1 infection and IFN-α stimulation assays were also performed. Genotyping was carried out on three independent European cohorts of HIV-1 exposed seronegative individuals (HESN) with different geographic origin and distinct exposure route. Variants were genotyped through direct sequencing. Genetic association analyses were performed by logistic regression and results from the three cohorts were combined using a random-effect meta-analysis; all analyses were performed using PLINK. For the HIV infection assay, PBMC from HESN subjects were separated on lymphocyte separation medium. After viability assessment, they were resuspended in a medium containing HIV-1Ba-L p24 viral input. After 7 days, supernatants were collected for p24 antigen ELISA analyses and absolute levels of p24 were measured. For IFN-α stimulation, freshly isolated PBMC from healthy controls were incubated with medium alone or with IFN-α. Cultured PBMC RNA was extracted and then reverse transcribed into first-strand cDNA. cDNA quantification was performed by a Real-Time PCR strategy. Results were presented as ratios between the target gene and the GAPDH housekeeping mRNA. RESULTS Evolutionary analysis can provide extremely relevant information not only on the evolutionary history of our genome, but also on the presence and location of functional genetic variants especially in relation to phenotypic diversity and, ultimately, human health. In the study of the brush border genes, results indicated pervasive selection in mammals and human populations, reflecting specific adaptation to specialized diets. Furthermore, I found that positively selected modern alleles predate the emergence of agriculture. In the second set of analyses I found that pervasive positive selection is driven by pressure related to immune response. Selection acted on functionally relevant protein regions (e.g.: regulatory regions, regions which confer antiviral activity) and residues. Interestingly, natural selection often targeted residues located in the same spatial position in different proteins. This is for example the case of sites detected in OAS1, OAS2 and MB21D1, which revealed parallel evolution. In the analyses of viral/protozoan/bacterial proteins and their host interactors, I found most of the positively selected sites within the region at the host-pathogen interface. These results epitomize the expectation under a genetic conflict scenario, whereby the host and the pathogen genes evolve within binding avoidance-binding seeking dynamics. Part of these sites had a role in host-pathogen binding affinity, some other adaptive changes most likely contributed to the shift to human hosts, and still other residues found to evolve under positive selection in viruses reside in antigenic determinants. Because antibody combinations are the most promising treatment strategies, these findings should pose a serious concern to their effectiveness in the long-term. CONCLUSIONS These works highlight the importance of evolutionary analysis. Specifically, I show that evolutionary studies can (i) provide information on the past adaptive events that shaped human-specific traits, (ii) identify functional regions and sites evolving under positive selection, (iii) predict host-pathogen interaction surfaces at the single amino acid resolution, (iv)provide valuable information on the molecular determinant underlying species-specific infection susceptibility and clarify the differential response to natural or synthetic molecules, (v) help identify the most likely reservoirs for zoonotic pathogens, (vi) explain changes in pathogen tropism and (vii) provide information on possible therapeutic targets (e.g. effectiveness of long-term treatment based on antibody combinations).