by Prof. Lemaitre

Laboratory

Fluorescent D-Amino Acids for Super-resolution Microscopy of the Bacterial Cell Wall

Juliette Griffie, Suliana Manley, Jiangtao Qiao, Luc Reymond, Ophélie Rutschmann, Faustine Ambroisine Charline Ryckebusch, Willi Leopold Stepp, Chen Zhang

Fluorescent D-amino acids (FDAAs) have previ-ously been developed to enable in situ highlighting of locations of bacterial cell wall growth. Most bacterial cells lie at the edge of the diffraction limit of visible light; thus, resolving the precise details of peptidoglycan (PG) biosynthesis requires super-resolution micros-copy after probe incorporation. Single molecule localization microscopy (SMLM) has stringent requirements on the fluorophore photophysical properties and therefore has remained challenging in this context. Here, we report the synthesis and characterization of new FDAAs compatible with one-step labeling and SMLM imaging. We demonstrate the incorporation of our probes and their utility for visualizing PG at the nanoscale in Gram-negative, Gram-positive, and mycobacteria species. This improved FDAA toolkit will endow researchers with a nanoscale perspective on the spatial distribution of PG biosynthesis for a broad range of bacterial species.

AMER CHEMICAL SOC2022

The role of the Nimrod proteins in the Drosophila immune system

Bianca Marie Petrignani

The use of tractable model organisms such as Drosophila melanogaster and the powerful genetic tools they offer has contributed significantly to recent advances in comprehension of innate immunity, notably phagocytosis. To date, several phagocytic transmembrane receptors, opsonins, and ligands have been discovered in Drosophila melanogaster. Many of them belong to the Nimrod family, which includes both secreted and transmembrane proteins containing several NIM repeats. Genetic and biochemical studies have revealed the roles of transmembrane Nimrod receptors such as Draper, Simu and Eater in phagocytosis. However, the function of the five secreted Nimrod members, the Nimrod B proteins, remains unstudied. The goals of my thesis were to characterize the function of the secreted Nimrod B proteins using newly generated CRISPR-Cas9 null mutations, focusing on NimB5, NimB4, and NimB1 genes. In this work, we first found that the secreted protein, NimB5, is produced in the fat body upon nutrient scarcity downstream of metabolic sensors and ecdysone signaling. We provide evidence that NimB5 binds to hemocytes to down-regulate their proliferation and adhesion. Blocking this signaling loop results in conditional lethality when larvae are raised on a poor diet due to excessive hemocyte numbers and insufficient energy storage. This pointed to a role of NimB5 in the allocation of resources to blood cells, tailoring the investment in the immune system to metabolic resource availability.In the second part of this thesis, we analyzed the function of NimB4 to reveal its crucial role in clearance of apoptotic cells. NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Our study points to a role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis. Finally, in the last part of the thesis, we present preliminary results on the role of NimB1 in the Drosophila immune system. NimB1 is expressed mainly in hemocytes and regulates hemocyte number. Additionally, NimB1 shares several characteristics with NimB4, notably the ability to bind to apoptotic cells. As such, we hypothesize that it could play a similar function in the phagocytosis of apoptotic cells. However, the exact role of NimB1 in phagocytosis is still unclear, and additional work is necessary to understand if NimB1 plays a role in the uptake of apoptotic cells or phagosome maturation. Collectively, this thesis has revealed that the NimB proteins play specific roles in efferocytosis or in blood cell number regulation. Thus, this work extended our knowledge on the Drosophila cellular immune response by providing new insight on the conserved Nimrod family of proteins.

EPFL2022

Cecropins contribute to Drosophila host defense against a subset of fungal and Gram-negative bacterial infection

Alexia Laurie Carboni, Mark Austin Hanson, Bruno Lemaitre

Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB, and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion Delta Cec(A-C) removing the whole cecropin locus. Using the Delta Cec(A-C) deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response. Delta Cec(A-C) flies were viable and resisted challenge with various microbes as wild-type. However, removing Delta Cec(A-C) in flies already lacking 10 other AMP genes revealed a role for Cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defense and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (Delta AMP14) that lacks 14 immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.

OXFORD UNIV PRESS INC2022

A systematic CRISPR approach to understanding the role of Drosophila antimicrobial peptides in immunity in vivo

Mark Austin Hanson

Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microbes. These short immune effectors are conserved in plants, animals, and fungi. Early work showed that AMPs killed bacteria in generalist fashions in vitro: i.e. AMPs that killed Escherichia coli also killed many Gram-negative bacteria when tested. At the genome level, AMP gene families rapidly expand or contract, which suggested single genes were unlikely to be important. At the signalling level, AMPs are induced as a suite of peptides by conserved NF-ÎºB immune pathways across organisms (e.g. Toll, Imd). Together these observations led to the assump-tion that individual genes contributed only small effects, and instead the cumulative cocktail of AMPs was key to a successful de-fence response. This idea was never robustly tested in vivo owing to technical limitations. In 2015, two studies stumbled onto re-markable effects of fruit fly immune effectors. In one case a polymorphism in a single AMP gene (Diptericin A or DptA) greatly af-fected the fly defence against a specific bacterium (Providencia rettgeri). In another case, deleting just the Bomanin gene family caused immune susceptibility mimicking loss of Toll signalling generally. The prevailing model of generalist AMP action was ill equipped to explain these findings.In my PhD, I have systematically deleted the AMP genes of fruit flies to clarify AMP defences in vivo. This confirmed AMPs can act in generalist or redundant fashions in some cases. However some AMP-pathogen interactions are remarkably specific. Deletion of just the Drosocin gene explains much of the susceptibility of NF-ÎºB/Imd immune deficient flies to Enterobacter cloacae infection. Meanwhile deletion of just the two Diptericins recapitulates the susceptibility of Imd mutants to P. rettgeri.Contrary to previous assumptions, our findings suggested AMPs are not simple generalist peptides. There are many more short peptide immune genes waiting to be characterized that may be relevant to specific infections. I next investigated three lesser-characterized genes: Baramicin A, DptB, and Drosocin. The Baramicin A gene encodes multiple products, including one peptide (IM22) that was first annotated in my study. I found BaraA is key to the fly defence against pathogenic fungi. Next, I dissected the roles of individual Diptericin genes. Surprisingly, DptB alone is required for survival after infection by a lab isolate of Acetobacter. Finally, I identified Drosocin as the source gene for IM7, a mystery peptide first detected in 1998. A polymorphism in IM7 previously obscured its identification. This polymorphism affects fly defence against Providencia burhodogranariea, where one immune-poor allele effectively rivals deletion of IM7 entirely against this microbe.These AMP-microbe interactions reveal that survival after infection can be mediated at the level of single AMP genes or even com-mon alleles of those genes. Contrary to previous assumptions, it appears the AMP response is composed of silver bullets, wooden stakes, and other specialized defence tools required to fight specific enemies. In this light, the diverse AMPs induced upon infection may be an evolutionary solution to optimize survival: general microbe patterns induce immune signalling, there is a need for timely production of antimicrobial peptides, and so a battery of antimicrobial peptides are produced immediately even though only a few are likely to be relevant for any given pa

EPFL2022

Gene-by-environment modulation of lifespan and weight gain in the murine BXD family

Johan Auwerx, Maroun Bou Sleiman, Pooja Jha, Evan Graehl Williams

How lifespan and body weight vary as a function of diet and genetic differences is not well understood. Here we quantify the impact of differences in diet on lifespan in a genetically diverse family of female mice, split into matched isogenic cohorts fed a low-fat chow diet (CD, n = 663) or a high-fat diet (HFD, n = 685). We further generate key metabolic data in a parallel cohort euthanized at four time points. HFD feeding shortens lifespan by 12%: equivalent to a decade in humans. Initial body weight and early weight gains account for longevity differences of roughly 4-6 days per gram. At 500 days, animals on a HFD typically gain four times as much weight as control, but variation in weight gain does not correlate with lifespan. Classic serum metabolites, often regarded as health biomarkers, are not necessarily strong predictors of longevity. Our data indicate that responses to a HFD are substantially modulated by gene-by-environment interactions, highlighting the importance of genetic variation in making accurate individualized dietary recommendations. Roy et al. quantify the impact of a high-fat diet across genetically diverse strains of mice, revealing a generally negative effect on lifespan but also a wide variability.

NATURE PORTFOLIO2021

A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila

Bruno Lemaitre, Florent François Masson, Bianca Marie Petrignani, Samuel Rommelaere

Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non-professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.

2021

Dual proteomics of Drosophila melanogaster hemolymph infected with the heritable endosymbiont Spiroplasma poulsonii

Bruno Lemaitre, Alice Marra, Florent François Masson, Samuel Rommelaere, Fanny Schüpfer

Insects are frequently infected with heritable bacterial endosymbionts. Endosymbionts have a dramatic impact on their host physiology and evolution. Their tissue distribution is variable with some species being housed intracellularly, some extracellularly and some having a mixed lifestyle. The impact of extracellular endosymbionts on the biofluids they colonize (e.g. insect hemolymph) is however difficult to appreciate because biofluid composition can depend on the contribution of numerous tissues. Here we investigate Drosophila hemolymph proteome changes in response to the infection with the endosymbiont Spiroplasma poulsonii. S. poulsonii inhabits the fly hemolymph and gets vertically transmitted over generations by hijacking the oogenesis in females. Using dual proteomics on infected hemolymph, we uncovered a weak, chronic activation of the Toll immune pathway by S. poulsonii that was previously undetected by transcriptomics-based approaches. Using Drosophila genetics, we also identified candidate proteins putatively involved in controlling S. poulsonii growth. Last, we also provide a deep proteome of S. poulsonii, which, in combination with previously published transcriptomics data, improves our understanding of the post-transcriptional regulations operating in this bacterium.

2021

Seascape Genomics of the Sugar Kelp Saccharina latissima along the North Eastern Atlantic Latitudinal Gradient

Chloé Christiane Laurence Jollivet, Stéphane Mauger

Temperature is one of the most important range-limiting factors for many seaweeds. Driven by the recent climatic changes, rapid northward shifts of species' distribution ranges can potentially modify the phylogeographic signature of Last Glacial Maximum. We explored this question in detail in the cold-tolerant kelp species Saccharina latissima, using microsatellites and double digest restriction site-associated DNA sequencing ( ddRAD-seq) derived single nucleotide polymorphisms (SNPs) to analyze the genetic diversity and structure in 11 sites spanning the entire European Atlantic latitudinal range of this species. In addition, we checked for statistical correlation between genetic marker allele frequencies and three environmental proxies (sea surface temperature, salinity, and water turbidity). Our findings revealed that genetic diversity was significantly higher for the northernmost locality (Spitsbergen) compared to the southern ones (Northern Iberia), which we discuss in light of the current state of knowledge on phylogeography of S. latissima and the potential influence of the recent climatic changes on the population structure of this species. Seven SNPs and 12 microsatellite alleles were found to be significantly associated with at least one of the three environmental variables. We speculate on the putative adaptive functions of the genes associated with the outlier markers and the importance of these markers for successful conservation and aquaculture strategies for S. latissima in this age of rapid global change.

MDPI2020

The role of immune effectors in controlling Drosophila-microbes interactions

Alice Marra

All live beings are in constant interaction with microorganisms that may be beneficial, deleterious or commensal. Insects in particular live in close contact with microorganisms. This is especially true for species, like the fruit fly Drosophila melanogaster that feed, lay eggs and develop on or close to decomposing organic matter. In contrast to vertebrates, insects did not evolve an adaptive immune system to combat pathogens selectively. They instead rely on surprisingly efficient innate defense mechanisms for the control and clearance of all microbes without any species-specific targeting. Innate immunity encompasses a wide range of mechanisms that rely on direct pathogen recognition and elimination. In addition, metabolic and behavioral responses also strongly affect the outcome of insect interactions with both pathogenic and non-pathogenic bacteria. Although the Drosophila immune system has been extensively described, little is known about the role of immune effectors in tolerating and controlling symbiotic microbes. For this reason, during my PhD studies, I investigated how Drosophila melanogaster immune effectors differentially interact with mutualistic symbionts. First, I investigated the role of some of the host antimicrobials, called antimicrobial peptides and lysozymes, in maintaining the homeostasis of the gut microbiota. I found that both antimicrobial peptides and lysozymes can actively regulate the gut microbiota composition and abundancy, especially during aging. In a second part of my thesis, I got interested in Spiroplasma, a heritable symbiotic bacterium that lives within the fly hemolymph. I characterized the role of the Drosophila iron transporter Transferrin 1 (Tsf1) during Spiroplasma-Drosophila symbiosis. I first showed that mutant flies for tsf1 have an impaired Spiroplasma load, due to iron relocation from the hemolymph to the fat body, where it becomes inaccessible for Spiroplasma. Furthermore, I demonstrated that Spiroplasma scavenges host iron only when it is bound to the protein, which points to Tsf1 and iron transport as a control mechanism for hemolymphatic symbionts. Collectively, my studies contribute to a better understanding of how the innate immune effectors interact with Drosophila microbial symbionts to both regulate and maintain stable, long-lasting, interactions.

EPFL2021