Publications related to Antimicrobial peptides

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

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

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

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

Much has been learned about the genetics of aging from studies in model organisms, but still little is known about naturally occurring alleles that contribute to variation in longevity. For example, analysis of mutants and transgenes has identified insulin signaling as a major regulator of longevity, yet whether standing variation in this pathway underlies microevolutionary changes in lifespan and correlated fitness traits remains largely unclear. Here, we have analyzed the genomes of a set of Drosophila melanogaster lines that have been maintained under direct selection for postponed reproduction and indirect selection for longevity, relative to unselected control lines, for over 35 years. We identified many candidate loci shaped by selection for longevity and late-life fertility, but - contrary to expectation - we did not find overrepresentation of canonical longevity genes. Instead, we found an enrichment of immunity genes, particularly in the Toll pathway, suggesting that evolutionary changes in immune function might underpin - in part - the evolution of late-life fertility and longevity. To test whether this genomic signature is causative, we performed functional experiments. In contrast to control flies, long-lived flies tended to downregulate the expression of antimicrobial peptides upon infection with age yet survived fungal, bacterial, and viral infections significantly better, consistent with alleviated immunosenescence. To examine whether genes of the Toll pathway directly affect longevity, we employed conditional knockdown using in vivo RNAi. In adults, RNAi against the Toll receptor extended lifespan, whereas silencing the pathway antagonist cactus--causing immune hyperactivation - dramatically shortened lifespan. Together, our results suggest that genetic changes in the age-dependent regulation of immune homeostasis might contribute to the evolution of longer life.

JOHN WILEY & SONS LTD2018

Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach

Camilla Carla Alma Ceroni, Anna Dostalova, Mark Austin Hanson, Bruno Lemaitre

Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilized CRISPR gene editing to delete all known immune-inducible AMPs of Drosophila, namely: 4 Attacins, 4 Cecropins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking all 14 AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, contributing either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.

Cambridge University Press2019

New insights on Drosophila antimicrobial peptide function in host defense and beyond

Mark Austin Hanson, Bruno Lemaitre

Since the first animal antimicrobial peptides (AMPs) were discovered in insects, Drosophila melanogaster has emerged as a powerful model for their characterization. Drosophila AMPs have been used extensively to monitor the activity of the Toll and Imd NF-κB pathways, but little was known of their precise functions. In this review, we summarize recent findings on the function of Drosophila AMPs not only for antimicrobial defense, but also in the gut, tumor control, and neurology. The integration of these new studies allows a new framework to be drawn that explains how AMPs can contribute simultaneously to microbe killing whilst also regulating important host cellular functions. These functions require that AMPs target not only negatively charged microbes but also aberrant host cells.

2019

Long-range activation of systemic immunity through peptidoglycan diffusion in Drosophila

Bruno Lemaitre, David Welchman

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient Relish(E20) flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that Relish(E20) flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila.

2009

The antimicrobial peptide defensin cooperates with tumour necrosis factor to drive tumour cell death in Drosophila

Anna Dostalova, Bruno Lemaitre

Antimicrobial peptides (AMPs) are small cationic molecules best known as mediators of the innate defence against microbial infection. While in vitro and ex vivo evidence suggest AMPs' capacity to kill cancer cells, in vivo demonstration of an anti-tumour role of endogenous AMPs is lacking. Using a Drosophila model of tumourigenesis, we demonstrate a role for the AMP Defensin in the control of tumour progression. Our results reveal that Tumour Necrosis Factor mediates exposure of phosphatidylserine (PS), which makes tumour cells selectively sensitive to the action of Defensin remotely secreted from tracheal and fat tissues. Defensin binds tumour cells in PS-enriched areas, provoking cell death and tumour regression. Altogether, our results provide the first in vivo demonstration for a role of an endogenous AMP as an anti-cancer agent, as well as a mechanism that explains tumour cell sensitivity to the action of AMPs.

ELIFE SCIENCES PUBLICATIONS LTD2019

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The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.

PUBLIC LIBRARY SCIENCE2021

Functional Analysis of PGRP-LA in Drosophila Immunity

Nichole Broderick, Bruno Lemaitre, Juan Camilo Paredes Escobar

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.

Public Library of Science2013