Biology subjects

Bussotti, G.

Publications and source records attributed to Bussotti, G..

9 recordsLinked to original sources

bioRxiv2023

A comparative genomics approach reveals a local genetic signature of Leishmania tropica in Morocco

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is an important health problem. Despite its high incidence in the country, the genomic heterogeneity of these parasites is still incompletely understood. In this study, we sequenced the genomes of 14 Moroccan isolates of L. tropica collected from confirmed cases of CL to investigate their genomic heterogeneity. Comparative genomics analyses were conducted by applying the recently established Genome Instability Pipeline (GIP), which allowed us to conduct phylogenomic and PCA analyses, and to assess genomic variations at the levels of the karyotype, gene copy number, and single nucleotide polymorphisms (SNPs). The results identified a core group of 12 isolates that were genetically highly related but evolutionarily distant to the reference genome as judged by the presence of over 100,000 SNPs, 75% of which were shared inside this core group. In addition, we identified two highly divergent strains, M3015 and Ltr_16, that were phylogenetically distinct between each other as well as to the core group and the reference genome. Read-depth analysis revealed important karyotypic variations across all isolates and uncovered important differences in gene copy number between the isolates of the core group and the L. tropica reference genome, as well as between the core group and M3015. In conclusion, our NGS results suggest the presence of a local SNP signature that distinguishes Moroccan L. tropica from other endemic regions and from the reference genome. These results pave the way for future research with a larger number of strains that will allow to correlate diverse phenotypes (resistance to treatments, virulence) and origins (geography, host species, year of isolation) to defined genomic signals that may represent interesting biomarker candidates.

genomics↗

bioRxiv2022

Leishmania allelic selection during experimental sand fly infection correlates with mutational signatures of oxidative DNA damage

Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. Survival of these parasites in their vertebrate and invertebrate hosts relies on their capacity to differentiate into distinct stages that are the result of a co-evolutionary process. These stages show in addition important phenotypic shifts that often impacts infection, affecting for example parasite pathogenicity, tissue tropism, or drug susceptibility. Despite their clinical relevance, the evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here we use Leishmania donovani as a trypanosomatid model pathogen to shed first light on parasite evolutionary adaptation during experimental sand fly infection. Applying a comparative genomics approach on hamster- isolated amastigotes and derived promastigotes before (input) and after (output) infection of Phlebotomus orientalis revealed a strong bottleneck effect on the parasite population as judged by principal component and phylogenetic analyses of input and output parasite DNA sequences. Despite random genetic drift caused by the bottleneck effect, our analyses revealed various genomic signals that seem under positive selection given their convergence between independent biological replicates. While no significant fluctuations in gene copy number were revealed between input and output parasites, convergent selection was observed for karyotype, haplotype and allelic changes during sand fly infection. Our analyses further uncovered signature mutations of oxidative DNA damage in the output parasite genomes, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a new model of Leishmania genomic adaptation during sand fly infection, where oxidative DNA damage and DNA repair processes drive haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei and Trypanosoma cruzi.

evolutionary biology↗

bioRxiv2021

GIP: An open-source computational pipeline for mapping genomic instability from protists to cancer cells

Genome instability has been recognized as a key driver for microbial and cancer adaptation and thus plays a central role in many human pathologies. Even though genome instability encompasses different types of genomic alterations, most available genome analysis software are limited to just one kind mutation or analytical step. To overcome this limitation and better understand the role of genetic changes in enhancing pathogenicity we established GIP, a novel, powerful bioinformatic pipeline for comparative genome analysis. Here we show its application to whole genome sequencing datasets of Leishmania, Plasmodium, Candida, and cancer. Applying GIP on available data sets validated our pipeline and demonstrated the power of our analysis tool to drive biological discovery. Applied to Plasmodium vivax genomes, our pipeline allowed us to uncover the convergent amplification of erythrocyte binding proteins and to identify a nullisomic strain. Re-analyzing genomes of drug adapted Candida albicans strains revealed correlated copy number variations of functionally related genes, strongly supporting a mechanism of epistatic adaptation through interacting gene-dosage changes. Our results illustrate how GIP can be used for the identification of aneuploidy, gene copy number variations, changes in nucleic acid sequences, and chromosomal rearrangements. Altogether, GIP can shed light on the genetic bases of cell adaptation and drive disease biomarker discovery. One Sentence SummaryGIP - a novel pipeline for detecting, comparing and visualizing genome instability.

bioinformatics↗

bioRxiv2021

Genome instability drives epistatic adaptation in the human pathogen Leishmania

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches we provide first evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover post-transcriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in snoRNA gene dosage with changes in rRNA 2-O-methylation and pseudouridylation, suggesting translational control is an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression, and genome-independent, compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery, and may be relevant to other fast evolving, eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer. One Sentence SummaryEpistatic interactions harness genome instability for Leishmania fitness gain.

genomics↗

bioRxiv2021

Post-transcriptional regulation of Leishmania fitness gain

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein abundance linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in abundance of flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

microbiology↗

bioRxiv2021

Three-dimensional interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art HPV integrated site capture (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a looping mechanism by which flanking host regions become amplified. Furthermore, using our HPV16-specific Region Capture Hi-C technique, we have determined that three-dimensional (3D) interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive host gene dysregulation through the disruption of local host:host 3D interactions known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a cancer-causing gene is not essential to influence such genes within an entire TAD and that these modifications to 3D interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

cancer biology↗

bioRxiv2020

The oxidative stress response and virulence of pathogenic Leptospira are controlled by the interplay of two peroxide stress regulators

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network. Author summaryLeptospirosis is a widespread infectious disease responsible for over one million of severe cases and 60 000 fatalities annually worldwide. This neglected and emerging disease has a worldwide distribution, but it mostly affects populations from developing countries in sub-tropical areas. The causative agents of leptospirosis are pathogenic bacterial Leptospira spp. There is a considerable deficit in our knowledge of these atypical bacteria, including their virulence mechanisms. In addition to the Leptospira PerRA regulator that represses defenses against peroxide, we have identified and characterized a second PerR regulator in pathogenic Leptospira species (PerRB) that participates in Leptospira tolerance to superoxide. Phenotypic and transcriptomic analyses of single PerRA and PerRB mutants suggest that the two PerRs fulfill distinct functions in the adaptation to oxidative stress. Concomitant inactivation of PerRA and PerRB resulted in a higher tolerance to both peroxide and superoxide. Moreover, the perRAperRB mutant lost its virulence. Major changes in gene expression, including a decreased expression of several virulence factors, were observed in the double perRAperRB mutant. Our study suggests that PerRA and PerRB cooperate to orchestrate a complex regulatory network involved in Leptospira virulence.

microbiology↗

bioRxiv2020

The adaptive transcriptional response of pathogenic Leptospira to peroxide reveals new defenses against infection-related oxidative stress

Pathogenic Leptospira spp. are the causative agents of the waterborne zoonotic disease leptospirosis. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen species (ROS). Withstanding ROS produced by the host innate immunity is an important strategy evolved by pathogenic Leptospira for persisting in and colonizing hosts. In L. interrogans, genes encoding defenses against ROS are repressed by the peroxide stress regulator, PerR. In this study, RNA sequencing was performed to characterize both the L. interrogans adaptive response to low and high concentrations of hydrogen peroxide and the PerR regulon. We showed that Leptospira solicit three main peroxidase machineries (catalase, cytochrome C peroxidase and peroxiredoxin) and heme to detoxify oxidants produced during a peroxide stress. In addition, canonical molecular chaperones of the heat shock response and DNA repair proteins from the SOS response were required for Leptospira recovering from oxidative damages. Determining the PerR regulon allowed to identify the PerR-dependent mechanisms of the peroxide adaptive response and has revealed a PerR-independent regulatory network involving other transcriptional regulators, two-component systems and sigma factors as well as non-coding RNAs that putatively orchestrate, in concert with PerR, this adaptive response. In addition, we have identified other PerR-regulated genes encoding a TonB-dependent transport system, a lipoprotein (LipL48) and a two-component system (VicKR) involved in Leptospira tolerance to superoxide and that could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide a comprehensive insight into the mechanisms required by pathogenic Leptospira to overcome infection-related oxidants during the arm race with a host. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen. Author summaryLeptospirosis is a zoonotic infectious disease responsible for over one million of severe cases and 60 000 fatalities annually worldwide. This neglected and emerging disease has a worldwide distribution, but it mostly affects populations from developing countries in sub-tropical areas. The causative agents of leptospirosis are pathogenic bacterial Leptospira spp. There is a considerable deficit in our knowledge of these atypical bacteria, including their virulence mechanisms. During infection, Leptospira are confronted with the deadly oxidants produced by the host tissues and immune response. Here, we have identified the cellular factors necessary for Leptospira to overcome the oxidative stress response. We found that Leptospira solicit peroxidases to detoxify oxidants as well as chaperones of the heat shock response and DNA repair proteins of the SOS response to recover from oxidative damage. Moreover, our study indicates that adaptation to oxidative stress is orchestrated by a regulatory network involving PerR and other transcriptional regulators, sigma factors, two component systems, and putative non-coding RNAs. These findings provide a comprehensive insight into the mechanisms required by pathogenic Leptospira to tolerate infection-related oxidants, helping identify novel virulence factors, developing new therapeutic targets and vaccines against leptospirosis.

microbiology↗

bioRxiv2019

Leishmania targets the macrophage epigenome and dampens the NF-κB/NLRP3- mediated inflammatory response

Aberrant macrophage activation during intracellular infection generates important immunopathologies that can cause severe human morbidity. A better understanding of microbial immune subversion strategies and macrophage phenotypic and functional responses is a prerequisite for the design of novel, host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response induced in primary macrophages infected by the human parasite Leishmania amazonensis that prevents NF-{kappa}B and NLRP3 inflammasome activation. This unusual subversion is characterized by respectively suppression and induction of activating and de-activating components of the NF-{kappa}B and NLRP3 pathways. This dichotomic modulation was associated with histone H3 hypoacetylation at promoters of NF-{kappa}B-related, pro-inflammatory genes. Our results reveal a novel Leishmania immune subversion strategy targeting host cell epigenetic regulation to modulate the macrophage phenotype. Modulation of the macrophage epigenetic landscape establishes conditions beneficial for intracellular parasite survival, and opens interesting new venues for host-directed, anti-microbial drug discovery.

cell biology↗