Antibiotics impact on human macrophages response : bedaquiline case

Les antibiotiques sont largement utilisés dans le traitement des infections bactériennes. Connus pour leur activité sur les bactéries, les antibiotiques peuvent également interférer avec le système immunitaire de l’hôte. L’objectif de cette thèse a été d’étudier l’impact de la bédaquiline (BDQ), un récent anti-tuberculeux, sur la réponse des macrophages humains. L’analyse de l’expression des gènes a pu révéler que la BDQ reprogramme les macrophages en augmentant l’expression des gènes liée aux fonctions bactéricides. Nous avons découvert que l’expression de 1495 gènes étaient différentiellement exprimées dans les macrophages infectés par Mycobacterium tuberculosis et traités par la BDQ, avec une régulation positive des gènes impliqués dans le métabolisme et l’activation des lysosomes et leur biogénèse. Le traitement par la BDQ a permis d’activer divers mécanismes de défense antimicrobiens, notamment la production d’oxyde nitrique, la fusion phagosome-lysosome et l’autophagie. Ces effets sont associés à l’activation du facteur de transcription EB, impliqué dans l’expression des gènes lysosomaux, entrainant une diminution de la survie intracellulaire de différentes espèces bactériennes naturellement resistante à la BDQ. Ainsi, la BDQ pourrait être utilisé comme composé HDT (host-directed therapy) contre les infections bactériennes.Antibiotics are widely used in the treatment of bacterial infections. Although known for theirmicrobicidal activity, antibiotics may also interfere with the host’s immune system. The aim of this thesis is to investigated the impact of a new anti-TB drug, bedaquiline, on the innate immune response of human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed macrophages into potent bactericidal phagocytes. We found that 1,495 genes were differentially expressed in M. tuberculosis-infected macrophages incubated with the drug, with an overrepresentation of genes involved in metabolism, lysosome biogenesis and activation. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including nitric oxide production, phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB (TFEB), involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ

Evolutionary insights into the emergence of virulent Leptospira spirochetes

Pathogenic Leptospira are spirochete bacteria which cause leptospirosis, a re-emerging zoonotic disease of global importance. Here, we use a recently described lineage of environmental-adapted leptospires, which are evolutionarily the closest relatives of the highly virulent Leptospira species, to explore the key phenotypic traits and genetic determinants of Leptospira virulence. Through a comprehensive approach integrating phylogenomic comparisons with in vitro and in vivo phenotyping studies, we show that the evolution towards pathogenicity is associated with both a decrease of the ability to survive in the environment and the acquisition of strategies that enable successful host colonization. This includes the evasion of the human complement system and the adaptations to avoid activation of the innate immune cells. Moreover, our analysis reveals specific genetic determinants that have undergone positive selection during the course of evolution in Leptospira, contributing directly to virulence and host adaptation as demonstrated by gain-of-function and knock-down studies. Taken together, our findings define a new vision on Leptospira pathogenicity, identifying virulence attributes associated with clinically relevant species, and provide insights into the evolution and emergence of these life-threatening pathogens.</br

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France

International audienceThe monitoring of SARS-CoV-2 RNA in sewage has been proposed as a simple and unbiased means of assessing epidemic evolution and the efficiency of the COVID-19 control measures. The past year has been marked by the emergence of variants that have led to a succession of epidemic waves. It thus appears that monitoring the presence of SARS-CoV-2 in wastewater alone is insufficient, and it may be important in the future to also monitor the evolution of these variants. We used a real-time RT-PCR screening test for variants in the wastewater of our city to assess the effectiveness of direct SARS-CoV-2 sequencing from the same wastewater. We compared the genome sequencing results obtained over the large RS network and the smaller B7 network with the different distributions of the variants observed by RT-PCR screening. The prevalence of the “UK variant” in the RS and B7 networks was estimated to be 70% and 8% using RT-PCR screening compared to 95% and 64% using genome sequencing, respectively. The latter values were close to the epidemiology observed in patients of the corresponding area, which were 91% and 58%, respectively. Genome sequencing in sewage identified SARS-CoV-2 of lineage B.1.525 in B7 at 27% (37% in patients), whereas it was completely missed by RT-PCR. We thus determined that direct sequencing makes it possible to observe, in wastewater, a distribution of the variants comparable to that revealed by genomic monitoring in patients and that this method is more accurate than RT-PCR. It also shows that, rather than a single large sample, it would be preferable to analyse several targeted samples if we want to more appropriately assess the geographical distribution of the different variants. In conclusion, this work supports the wider surveillance of SARS-CoV-2 variants in wastewater by genome sequencing and targeting small areas on the condition of having a sequencing capacity and, when this is not the case, to developing more precise screening tests based on the multiplexed detection of the mutations of interest

Alive pathogenic and saprophytic leptospires enter and exit human and mouse macrophages with no intracellular replication

International audienceLeptospira interrogans are pathogenic bacteria responsible for leptospirosis, a zoonosis impacting 1 million people per year worldwide. Leptospires can infect all vertebrates, but not all hosts develop similar symptoms. Human and cattle may suffer from mild to acute illness and are therefore considered as sensitive to leptospirosis. In contrast, mice and rats remain asymptomatic upon infection, although they get chronically colonized in their kidneys. Upon infection, leptospires are stealth pathogen that partially escape the recognition by the host innate immune system. Although leptospires are mainly extracellular bacteria, it was suggested that they could also replicate within macrophages. However, contradictory data in the current literature led us to reevaluate these findings. Using a gentamicin protection assay coupled to high content (HC) microscopy, we observed that leptospires were internalized in vivo upon peritoneal infection of C57BL/6J mice. Additionally, three different serotypes of pathogenic L. interrogans and the saprophytic L. biflexa actively infected both human (PMA differentiated) THP1 and mouse RAW264.7 macrophage cell lines. Next, we assessed the intracellular fate of leptospires using bioluminescent strains, and we observed a drastic reduction in the leptospiral intracellular load between 3h and 6h post-infection, suggesting that leptospires do not replicate within these cells. Surprisingly, the classical macrophage microbicidal mechanisms (phagocytosis, autophagy, TLR mediated ROS and RNS production) were not responsible for the observed decrease. Finally, we demonstrated that the reduction of the intracellular load was associated with an increase of the bacteria in the supernatant, suggesting that leptospires exit both human and murine macrophages. Overall, our study reevaluated the intracellular fate of leptospires and favors an active entrance followed by a rapid exit, suggesting that leptospires do not have an intracellular lifestyle in macrophages

Viral RNA in City Wastewater as a Key Indicator of COVID-19 Recrudescence and Containment Measures Effectiveness

International audienceIn recent years, and more specifically at the beginning of the COVID-19 crisis, wastewater surveillance has been proposed as a tool to monitor the epidemiology of human viral infections. In the present work, from July to December 2020, the number of copies of SARS-CoV-2 RNA in Marseille’s wastewater was correlated with the number of new positive cases diagnosed in our Institute of Infectious Disease, which tested about 20% of the city’s population. Number of positive cases and number of copies of SARS-CoV-2 RNA in wastewater were significantly correlated ( p = 0.013). During the great epidemic peak, from October to December 2020, the curves of virus in the sewers and the curves of positive diagnoses were perfectly superposed. During the summer period, the superposition of curves was less evident as subject to many confounding factors that were discussed. We also tried to correlate the effect of viral circulation in wastewater with containment measures, probably the most unbiased correlation on their potential inflection effect of epidemic curves. Not only is this correlation not obvious, but it also clearly appears that the drop in cases as well as the drop in the viral load in the sewers occur before the containment measures. In fact, this suggests that there are factors that initiate the end of the epidemic peak independently of the containment measure. These factors will therefore need to be explored more deeply in the future

Inter-species Transcriptomic Analysis Reveals a Constitutive Adaptation Against Oxidative Stress for the Highly Virulent <i>Leptospira</i> Species

International audienceTranscriptomic analyses across large scales of evolutionary distance have great potential to shed light on regulatory evolution but are complicated by difficulties in establishing orthology and limited availability of accessible software. We introduce here a method and a graphical user interface wrapper, called Annotator-RNAtor, for performing interspecies transcriptomic analysis and studying intragenus evolution. The pipeline uses third-party software to infer homologous genes in various species and highlight differences in the expression of the core-genes. To illustrate the methodology and demonstrate its usefulness, we focus on the emergence of the highly virulent Leptospira subclade known as P1+, which includes the causative agents of leptospirosis. Here, we expand on the genomic study through the comparison of transcriptomes between species from P1+ and their related P1- counterparts (low-virulent pathogens). In doing so, we shed light on differentially expressed pathways and focused on describing a specific example of adaptation based on a differential expression of PerRA-controlled genes. We showed that P1+ species exhibit higher expression of the katE gene, a well-known virulence determinant in pathogenic Leptospira species correlated with greater tolerance to peroxide. Switching PerRA alleles between P1+ and P1- species demonstrated that the lower repression of katE and greater tolerance to peroxide in P1+ species was solely controlled by PerRA and partly caused by a PerRA amino-acid permutation. Overall, these results demonstrate the strategic fit of the methodology and its ability to decipher adaptive transcriptomic changes, not observable by comparative genome analysis, that may have been implicated in the emergence of these pathogens

Lipid A structural diversity among members of the genus Leptospira

International audienceLipid A is the hydrophobic component of bacterial lipopolysaccharide and an activator of the host immune system. Bacteria modify their lipid A structure to adapt to the surrounding environment and, in some cases, to evade recognition by host immune cells. In this study, lipid A structural diversity within the Leptospira genus was explored. The individual Leptospira species have dramatically different pathogenic potential that ranges from non-infectious to life-threatening disease (leptospirosis). Ten distinct lipid A profiles, denoted L1-L10, were discovered across 31 Leptospira reference species, laying a foundation for lipid A-based molecular typing. Tandem MS analysis revealed structural features of Leptospira membrane lipids that might alter recognition of its lipid A by the host innate immune receptors. Results of this study will aid development of strategies to improve diagnosis and surveillance of leptospirosis, as well as guide functional studies on Leptospira lipid A activity

Tri-mannose grafting of chitosan nanocarriers remodels the macrophage response to bacterial infection

International audienceBackground: Infectious diseases are still a leading cause of death and, with the emergence of drug resistance, pose a great threat to human health. New drugs and strategies are thus urgently needed to improve treatment efficacy and limit drug-associated side effects. Nanotechnology-based drug delivery systems are promising approaches, offering hope in the fight against drug resistant bacteria. However, how nanocarriers influence the response of innate immune cells to bacterial infection is mostly unknown. Results: Here, we used Mycobacterium tuberculosis as a model of bacterial infection to examine the impact of man-nose functionalization of chitosan nanocarriers (CS-NCs) on the human macrophage response. Both ungrafted and grafted CS-NCs were similarly internalized by macrophages, via an actin cytoskeleton-dependent process. Although tri-mannose ligands did not modify the capacity of CS-NCs to escape lysosomal degradation, they profoundly remod-eled the response of M. tuberculosis-infected macrophages. mRNA sequencing showed nearly 900 genes to be differentially expressed due to tri-mannose grafting. Unexpectedly, the set of modulated genes was enriched for pathways involved in cell metabolism, particularly oxidative phosphorylation and sugar metabolism. Conclusions: The ability to modulate cell metabolism by grafting ligands at the surface of nanoparticles may thus be a promising strategy to reprogram immune cells and improve the efficacy of encapsulated drugs

The PACASurvE laboratory network for real-time infection surveillance and alert at a regional scale

International audienceirregular ratios. Seven models issued from time series analysis and three ensemble stacking models (average, convex and linear stacking) were used to describe and forecast CPE episodes. The model with the best forecasting's quality was then trained on all available data (2010-2016) and used to predict CPE episodes over 2017-2020. Results: Over 2010-2016, 3,559 CPE episodes were observed in France. Compared to the average yearly trend, we observed a 30% increase in the number of CPE episodes in September and October. On the opposite, a decrease of 20% was noticed in February compared to other months. We also noticed a 1-month lagged seasonality of non-imported episodes compared to imported ones. The number of non-imported episodes appeared to grow faster than imported ones starting from 2014. Average stacking gave the best forecasts and predicted an increase over 2017-2020 with a peak up to 345 CPE episodes (95% PI [124-1,158], 80% PI [171-742]) in September 2020. Conclusions: The number of CPE episodes is predicted to rise in the next years in France because of non-imported episodes. These results could help public health authorities in the definition and evaluation of new containment strategies. Key messages: Time series modeling predicts an increase in the number of CPE episodes in France in the next few years with a quicker rise of non-imported episodes. An increase of 30% in the number of CPE episodes was observed in September and October with a 1-month lagged seasonality impact of non-imported episodes compared to imported one

The PACASurvE laboratory network for real-time infection surveillance and alert at a regional scale

International audienceirregular ratios. Seven models issued from time series analysis and three ensemble stacking models (average, convex and linear stacking) were used to describe and forecast CPE episodes. The model with the best forecasting's quality was then trained on all available data (2010-2016) and used to predict CPE episodes over 2017-2020. Results: Over 2010-2016, 3,559 CPE episodes were observed in France. Compared to the average yearly trend, we observed a 30% increase in the number of CPE episodes in September and October. On the opposite, a decrease of 20% was noticed in February compared to other months. We also noticed a 1-month lagged seasonality of non-imported episodes compared to imported ones. The number of non-imported episodes appeared to grow faster than imported ones starting from 2014. Average stacking gave the best forecasts and predicted an increase over 2017-2020 with a peak up to 345 CPE episodes (95% PI [124-1,158], 80% PI [171-742]) in September 2020. Conclusions: The number of CPE episodes is predicted to rise in the next years in France because of non-imported episodes. These results could help public health authorities in the definition and evaluation of new containment strategies. Key messages: Time series modeling predicts an increase in the number of CPE episodes in France in the next few years with a quicker rise of non-imported episodes. An increase of 30% in the number of CPE episodes was observed in September and October with a 1-month lagged seasonality impact of non-imported episodes compared to imported one