Biology subjects

Labroussaa, F.

Publications and source records attributed to Labroussaa, F..

7 recordsLinked to original sources

bioRxiv2023

Temperature effects on the magnitude of the bovine ex vivo immune response towards Mycoplasmopsis bovis

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of {gamma}{delta} T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host-pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12-14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, {gamma}{delta} T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-{gamma} secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host-pathogen interactions at different temperatures, including commonly used 37 {degrees}C, ruminant body temperature (38-38.5 {degrees}C) and fever ([≥]39.5 {degrees}C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 {degrees}C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response.

immunology↗

bioRxiv2022

Impaired immune response drives age-dependent severity of COVID-19

SARS-CoV-2 is a highly contagious respiratory virus and the causative agent for COVID-19. The severity of disease varies from mildly symptomatic to lethal and shows an extraordinary correlation with increasing age, which represents the major risk factor for severe COVID-191. However, the precise pathomechanisms leading to aggravated disease in the elderly are currently unknown. Delayed and insufficient antiviral immune responses early after infection as well as dysregulated and overshooting immunopathological processes late during disease were suggested as possible mechanisms. Here we show that the age-dependent increase of COVID-19 severity is caused by the disruption of a timely and well-coordinated innate and adaptive immune response due to impaired interferon (IFN) responses. To overcome the limitations of mechanistic studies in humans, we generated a mouse model for severe COVID-19 and compared the kinetics of the immune responses in adult and aged mice at different time points after infection. Aggravated disease in aged mice was characterized by a diminished IFN-{gamma} response and excessive virus replication. Accordingly, adult IFN-{gamma} receptor-deficient mice phenocopied the age-related disease severity and supplementation of IFN-{gamma} reversed the increased disease susceptibility of aged mice. Mimicking impaired type I IFN immunity in adult and aged mice, a second major risk factor for severe COVID-192-4, we found that therapeutic treatment with IFN-{lambda} in adult and a combinatorial treatment with IFN-{gamma} and IFN-{lambda} in aged Ifnar1-/-mice was highly efficient in protecting against severe disease. Our findings provide an explanation for the age-dependent disease severity of COVID-19 and clarify the nonredundant antiviral functions of type I, II and III IFNs during SARS-CoV-2 infection in an age-dependent manner. Based on our data, we suggest that highly vulnerable individuals combining both risk factors, advanced age and an impaired type I IFN immunity, may greatly benefit from immunotherapy combining IFN-{gamma} and IFN-{lambda}.

immunology↗

bioRxiv2021

Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, but not B.1.351, in animal models

Emerging variants of concern (VOCs) drive the SARS-CoV-2 pandemic. We assessed VOC B.1.1.7, now prevalent in several countries, and VOC B.1.351, representing the greatest threat to populations with immunity to the early SARS-CoV-2 progenitors. B.1.1.7 showed a clear fitness advantage over the progenitor variant (wt-S614G) in ferrets and two mouse models, where the substitutions in the spike glycoprotein were major drivers for fitness advantage. In the "superspreader" hamster model, B.1.1.7 and wt-S614G had comparable fitness, whereas B.1.351 was outcompeted. The VOCs had similar replication kinetics as compared to wt-S614G in human airway epithelial cultures. Our study highlights the importance of using multiple models for complete fitness characterization of VOCs and demonstrates adaptation of B.1.1.7 towards increased upper respiratory tract replication and enhanced transmission in vivo. Summary sentenceB.1.1.7 VOC outcompetes progenitor SARS-CoV-2 in upper respiratory tract replication competition in vivo.

microbiology↗

bioRxiv2021

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They lack a cell wall and have been suggested to have progressed through reductive evolution from phylogenetically closely related Clostridia. They are known to colonize the respiratory tract or the urogenital tract among other organs and can cause chronic and subclinical diseases associated with long persistence of the causative agent. Toxin-antitoxin systems (TAS) are genetic elements that have been described for several respiratory and urogenital pathogens as well as for Clostridia, but never for pathogenic mycoplasmas. Here we describe for the first-time different types of TAS in a Mycoplasma pathogen, namely M. mycoides subsp. capri. We identified candidate TAS in silico via TASmania database. Two candidate TAS identified in silico and another candidate TAS suggested in a minimal cell based on transposon mutagenesis were systematically tested for their functionality in hosts with different phylogenetic distance using heterologous expression. Phylogenetic distance of the host used for heterologous expression influenced the outcome of the functional testing. We corroborated functionality of the three candidate TAS in Mycoplasma capricolum subsp. capricolum. Moreover, we confirmed transcription and translation of molecules of the TAS investigated during in vitro growth. We sequence analyzed 15 genomes of M. mycoides subsp. capri and revealed an unequal distribution of the TAS studied pointing towards dynamic gain and loss of TAS within the species. Author summaryMycoplasmas have a minimal genome and have never been shown to possess TAS. In this work we showed the presence of different functional TAS systems in Mycoplasma mycoides subsp. capri, a caprine pathogen for the first time. Sequence analysis of a number of Mycoplasma mycoides subsp. capri strains revealed a plasticity of the genome with respect to TAS carriage. This work paves the way to investigate the biological role of TAS (e.g. persistence, stress tolerance) during infection using mycoplasmas as a simple model organism. Since most mycoplasmas lack classical virulence factors such as exotoxins and go into a kind of stealth mode to evade the immune system, TAS are likely to contribute to the parasitic lifestyle of mycoplasmas and should be investigated in that respect. The availability of synthetic genomics tools to modify a range of Mycoplasma pathogens and well-established challenge models for the latter mycoplasmas will foster future research on TAS in mycoplasmas.

microbiology↗

bioRxiv2020

SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic1. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

microbiology↗

bioRxiv2020

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability1-3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.

microbiology↗

bioRxiv2019

Removal of a subset of non-essential genes fully attenuates a highly virulent Mycoplasma strain

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects and plants. Here, we demonstrate that highly virulent Mycoplasma mycoides subspecies capri (Mmc) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately ten percent of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually nonessential in the JCVI-Syn3.0 minimal cell, were excised from the genome. In vitro characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though ten percent of the genome content were removed. A novel in vivo challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reaching endpoint criteria within seven days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.\n\nIMPORTANCEMembers of the Mycoplasma mycoides cluster cause important animal plaques in Africa and Asia, which impact animal welfare, provision of food and the life of thousands of small-scale farmers. We applied synthetic biology tools to Mycoplasma mycoides in order to design and create a fully attenuated Mycoplasma strain that was subsequently confirmed in vivo using a novel caprine infection model. This is the first time that a Mycoplasma mutant developed by applying synthetic biology tools has been tested in vivo to pin point candidate virulence traits. The mutant strain is similar to \"apathogenic E. coli K12\" strains that boosted the research on host-pathogen interactions for the genus Escherichia and other bacterial genera.

microbiology↗