Biology subjects

Lemaitre, B.

Publications and source records attributed to Lemaitre, B..

At least 19 recordsLinked to original sources

bioRxiv2026

The NimB2 opsonin promotes S. aureus recognition by macrophages in Drosophila melanogaster

Phagocytosis is a conserved effector process of innate immunity that enables specialized immune cells to detect, engulf, and degrade invading microbes as well as dying cells and cellular debris. Soluble opsonins enhance this process by coating target surfaces and promoting their recognition and uptake by phagocytic cells. In Drosophila, many phagocytic receptors of the Nimrod family have been characterized, but opsonins remain comparatively poorly understood. Here, we identified the secreted Nimrod protein NimB2 as an insect opsonin that promotes the clearance of Staphylococcus aureus. NimB2 is produced by the fat body, secreted into the hemolymph, and required for resistance to S. aureus infection. NimB2 null mutants showed reduced phagocytosis of S. aureus while maintaining Toll dependent antimicrobial peptide expression. NimB2 promotes S. aureus binding to plasmatocytes (Drosophila macrophages). Using binding assays and bacterial cell wall mutants, we found that NimB2 recognizes a lipoteichoic acid (LTA) dependent determinant on the S. aureus surface and coats the bacterium. We further show that this coating enables efficient recognition by hemocytes via the phagocytic receptor Eater, which is necessary for NimB2 dependent binding. Moreover, Eater overexpression enhances NimB2 mediated association of S. aureus with hemocytes. Together, our results establish NimB2 as an insect opsonin that links a specific bacterial ligand to a phagocytic receptor.

immunology↗

bioRxiv2026

The phosphatidylserine-binding proteins Turandots protect the peripheral nervous system from antimicrobial peptide toxicity

Inflammation increases with aging and contributes to neurodegeneration, yet the principles that determine how immune effectors target host neural tissue remain poorly understood. Antimicrobial peptides (AMPs) are central components of innate immune defenses strongly induced upon infection and upon aging. Studies have shown that AMPs can exhibit cytotoxicity toward host cells, pointing to a role in neurodegeneration. We show that cationic AMPs selectively bind and damage motoneurons that expose phosphatidylserine (PS), an anionic phospholipid normally restricted to the inner leaflet of the plasma membrane. Both infection and aging increase neuronal PS exposure alongside AMP expression. AMP binding occurs in a PS-dependent manner, leading to synaptic bouton fragmentation, accelerated neuronal aging, and locomotor decline. This toxicity is prevented in Drosophila by Turandot proteins, which reduce AMP-PS interactions on motoneurons. Together, our findings define a molecular mechanism underlying neuronal susceptibility to immunopathology and a set of proteins with neuroprotective potential. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/721952v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@ca817aorg.highwire.dtl.DTLVardef@fa8cb0org.highwire.dtl.DTLVardef@12a8bd4org.highwire.dtl.DTLVardef@423907_HPS_FORMAT_FIGEXP M_FIG This study shows that infection or dysbiosis in Drosophila can simultaneously induce antimicrobial peptide expression while promoting phosphatidylserine (PS) exposure on neurons at the neuromuscular junction. Cationic antimicrobial peptides contribute to neurodegeneration by binding to neurons that expose negatively charged phospholipids such as PS. In Drosophila, a family of secreted peptides, the Turandot proteins, can protect the peripheral nervous system by binding to PS-exposed membranes. Together, these findings reveal a role for antimicrobial peptides, a key component of innate immunity, in promoting neurodegeneration as well as a potential protective mechanism by PS masking agent. C_FIG

immunology↗

bioRxiv2025

The serine protease homolog Skanda modulates Toll-Phenoloxidase-mediated immunity in Drosophila

Extracellular serine protease (SP) cascades are central regulators of insect innate immunity. These cascades are negatively controlled by serine protease inhibitors (serpins) and fine-tuned by serine protease homologs (SPHs), which resemble SPs but lack catalytic activity. In Drosophila, a key SP cascade--the Toll-phenoloxidase (PO) pathway--governs both the melanization response and Toll-dependent antimicrobial peptide production. This cascade is triggered by secreted pattern-recognition receptors or microbial proteases and converges on two clip-domain SPs, Persephone and Hayan, which activate the Toll ligand Spatzle via the Spatzle-Processing Enzyme (SPE) and process prophenoloxidases. Here, we characterize the SPH Skanda and uncover its role in the Toll-PO cascade. skanda is genomically clustered with hayan and persephone and is transcriptionally induced upon infection. Skanda is unusual among SPHs, containing a long serine/threonine-rich region, two clip domains, and atypical disulfide bonds. Skanda is unstable and subject to cleavage by Grass. Functional assays show that Skanda dampens activation of Hayan, and to a lesser extent Persephone, within the Toll-PO cascade. Notably, skanda-deficient flies are highly susceptible to Staphylococcus aureus despite displaying normal Toll signaling and cuticular melanization. Moreover, compound mutants lacking two members of the hayan-psh-skanda cluster reveal a hidden contribution of Skanda to Toll activation in the absence of Persephone. Together, our results identify Skanda as a modulatory SPH that fine-tunes Toll pathway activity in concert with Persephone and Hayan.

immunology↗

bioRxiv2025

A Controlled Test of Risk-Dependent Immune Investment in a Clonal Ant

Biological systems can benefit from distributing defensive investment unevenly, concentrating protection in parts of the system that face higher risk or hold greater strategic value. In social groups, individuals often face asymmetric infection risks based on their behavioural roles, raising the possibility that immune defences are plastically adjusted according to risk. In social insects, this idea has led to the hypothesis that individuals performing high-risk tasks, such as foraging, should invest more in constitutive immune defences to reduce transmission within the colony. However, testing this hypothesis has been hindered by confounding effects of genotype, age, and infection history. We use the clonal raider ant Ooceraea biroi to overcome these limitations. In this system, spontaneous behavioural specialisation between genetically identical, age-matched individuals creates variation in infection risk, while controlling for other sources of variation. We first annotated the O. biroi immune gene repertoire and sequenced the transcriptomes of 77 individuals that showed considerable behavioural variation. We then combined fine-scale individual behavioural tracking with three complementary measures of immune investment: immune-related gene expression, antibacterial activity, and survival following infection. Despite behavioural specialisation, we find no evidence that individuals engaging in higher-risk behaviours invest more in constitutive immune defences. These results contradict a long-standing hypothesis in the field and suggest limits to plastic immune allocation based on infection risk in social insect colonies.

ecology↗

bioRxiv2025

Drosophila host defense mechanisms against filamentous fungal pathogens with diverse lifestyles

Entomopathogenic fungi serve as powerful regulators of insect populations in nature. However, how immune effectors combat fungal pathogens remains incompletely understood. We employ Drosophila melanogaster as a genetically tractable model to dissect immune defense mechanisms against diverse fungal pathogens. We show that the Toll pathway is the key determinant of immunity against all species tested regardless of their ecological strategy, primarily through resistance mechanisms that limit fungal proliferation. In addition, melanization, but not phagocytosis or the Imd pathway, also has a role in limiting fungal entry and proliferation. Additionally, we show that fungal protease detection by Persephone has a quantitatively more critical role than the glucan sensor GNBP3 in the activation of the Toll pathway upon fungal infection. Our study also reveals that the fly-obligate fungus Entomophthora muscae employs a vegetative development strategy to hide from the host immune response. These findings reveal that Drosophila immune mechanisms effectively defend against a broad range of fungal pathogens, while highlighting striking adaptations to overcome these defenses in highly specialized fungal pathogens such as E. muscae.

immunology↗

bioRxiv2025

Reproducibility of Scientific Claims in Drosophila Immunity: A Retrospective Analysis of 400 Publications

Drosophila immunity has been the focus of intense study and has impacted other research fields including innate immunity and agriculturally or epidemiologically relevant investigations of insect pests and vectors. Unsurprisingly for such a large body of work, some published results were later found to be irreplicable. Although some results have been contradicted in the literature, many have no published follow-up, either due to a lack of research or low motivation to publish negative or contradictory results. We have addressed this by performing a reproducibility project that analyses the conceptual replicability of claims from articles published on Drosophila immunity before 2011. To assess replicability, we extracted claims from 400 articles on the Drosophila immune response to bacteria and fungi and performed preliminary verification by comparing these claims to other published literature in the field. Using alternative approaches, we also experimentally tested some unchallenged claims, with no published follow-up. The intent of this analysis was to centralize evidence on insights and findings to improve clarity for scientists that may base research programs on these data. Although the aim of the ReproSci project is to assess the replicability of claims made in articles published in the field of Drosophila immunity, it is in no way an assessment of the scientific value of the research. All our data are published on a publicly available website associated with this article (https://ReproSci.epfl.ch/) that encourages community participation. This article provides a short summary of claims that were found to have contradictory evidence, which may help the community to assess past findings on Drosophila immunity and improve clarity going forward. Statistical analysis of this reproducibility study and metascience insights obtained from this approach are discussed in a companion article.

genetics↗

bioRxiv2025

A retrospective analysis of 400 publications reveals patterns of irreproducibility across an entire life sciences research field

The ReproSci project retrospectively analyzed the conceptual replicability of 1006 claims from 400 papers published between 1959 and 2011 in the field of Drosophila immunity. This project attempts to provide a comprehensive assessment, 14 years later, of the replicability of nearly all publications across an entire scientific community in experimental life sciences. We found that 61% of claims were verified, while only 7% were directly challenged (not replicable), a replicability rate higher than previous assessments. Notably, 24% of claims had never been independently tested and remain unchallenged. We performed experimental validations of a selection of 45 unchallenged claim, that revealed that a significant fraction (38/45) of them is in fact non-replicable. We also found that high-impact journals and top-ranked institutions are more likely to publish challenged claims. In line with the reproducibility crisis narrative, the rates of both challenged and unchallenged claims increased over time, especially as the field gained popularity. We characterized the uneven distribution of irreplicability among first and last authors. Surprisingly, irreplicability rates were similar between PhD students and postdocs, and did not decrease with experience or publication count. However, group leaders, who had prior experience as first authors in another Drosophila immunity team, had lower irreplicability rates, underscoring the importance of early-career training. Finally, authors with a more exploratory, short-term engagement with the field exhibited slightly higher rates of challenged claims and a markedly higher proportion of unchallenged ones. While limited by its reliance on published literature, this systematic, field-wide retrospective study offers meaningful perspective into the ongoing discussion on reproducibility in experimental life sciences.

scientific communication and education↗

bioRxiv2025

Functional Characterization of Paillotin: An Immune Peptide Regulated by the Imd Pathway with Pathogen-Specific Roles in Drosophila Immunity

Insects, such as Drosophila melanogaster, rely on innate immune defenses to combat microbial threats. Antimicrobial peptides (AMPs) play an important role in limiting pathogen entry and colonization. Despite intensive research into the regulation and biochemical properties of AMPs, their exact significance in vivo has remained uncertain due to the challenges of mutating small genes. Fortunately, recent technologies have enabled the mutation of individual AMP genes, overcome previous obstacles, and opened new avenues for research. In this study, we characterized one novel host-defense peptide, Paillotin (IM18, CG33706), using loss-of-function mutants. Paillotin is an ancient host defense peptide of Diptera, regulated by the Imd pathway. Loss of Paillotin does not impact the activity of either the Imd or Toll pathways. Importantly, we found that Paillotin mutants are viable but exhibit increased susceptibility to specific infections, particularly Providencia burhodogranariea. Paillotin was further found to contribute synergistically to defense against P. burhodogranariea when combined with other AMPs. However, we did not detect direct microbicidal activity of Paillotin in vitro in our hands. Taken together, our findings identify Paillotin as a novel host defense peptide acting downstream of Imd signaling, advancing our understanding of the Drosophila antimicrobial response.

immunology↗

bioRxiv2025

Layers of immunity: Deconstructing the Drosophila effector response

The host innate immune response relies on the cooperation of multiple defence modules. In insects and other arthropods, which have only innate immune mechanisms, four main immune-specific modules have been described in the defence against microbial invaders: the Toll pathway, the Imd pathway, the melanization response, and phagocytosis by plasmatocytes. Our present understanding of their relative importance remains fragmented as their contribution to host defence has never been simultaneously assessed across a large panel of pathogens. Here, we have taken advantage of newly-described immune mutants in a controlled genetic background to systematically delete these four immune modules individually, in pairs, or even all four simultaneously. Surprisingly, immune deficient flies simultaneously deficient in all four immune modules are viable, homozygous fertile, and display no overt morphological defects, suggesting these immune mechanisms are not strictly required for organismal development. With this new set of lines, we assessed the contribution of each module individually and collectively to host defense against five viruses, three fungi, eight Gram-positive bacteria, and eight Gram-negative bacteria. Our findings show that these four modules largely function independently and additively in host defense, although synergistic effects can occur for select pairs of modules. Our study confirmed the importance of the Imd pathway against Gram-negative bacteria and the Toll pathway against Gram-positive bacteria and fungi, largely via the induction of effectors such as antimicrobial peptides (AMPs) and Bomanins (Bom), but also reveals the important role of melanization against viruses, and the contribution of phagocytosis against various germs. Additionally, by examining microbial load kinetics in different mutants, we provide insight into how these modules contribute to tolerance or resistance against specific microbes. Our study provides insights into the architecture of the Drosophila immune system, revealing differential requirements of immune modules according to each pathogen. The set of immune deficient lines provided here offer tools to better assess the role of these immune modules in host defense.

immunology↗

bioRxiv2024

An updated proteomic analysis of Drosophila hemolymph after bacterial infection

Using an in-depth Mass Spectrometry based proteomics approach, we provide a comprehensivecharacterization of the hemolymphatic proteome of adult flies upon bacterial infection. We detected and quantified changes in abundance of several known immune regulators and effectors, including multiple antimicrobial peptides, peptidoglycan-binding proteins and serine proteases. Comparison to previously published transcriptomic analyses reveals a partial overlap with our dataset, indicating that many proteins released into the hemolymph upon infection may not be regulated at the transcript level. Among them, we identify a set of muscle-derived proteins released into the hemolymph upon infection. Finally, our analysis reveals that infection induces major changes in the abundance of proteins associated with mitochondrial respiration. This study uncovers a large number of previously undescribed proteins potentially involved in the immune response.

immunology↗

bioRxiv2024

Gut Commensal Bacteria-Derived Methionine is Required for Host Reproduction by Modulating RNA m6A Methylation of the Insulin Receptor

Gut commensal bacteria promote host reproduction by modulating metabolism and nutrition, yet the molecular mechanisms by which microbes modulate reproduction remain unclear. Here, we show that gut commensal bacteria promote host reproduction by providing amino-acid methionine, which controls the RNA m6A modification level of insulin receptor (InR) in the ovary of the invasive insect Bactrocera dorsalis. RNA m6A levels and S-adenosyl-methionine (SAM) titers in the ovaries were sharply reduced in antibiotic treated B. dorsalis compared with untreated insects, resulting in arrested ovarian development and decreased fecundity. The intestinal commensal bacteria Enterobacter hormaechei or the E. hormaechei-derived metabolite methionine restored the decreased RNA m6A level and the reproductive defects. Notably, knockdown of METTL3 and METTL14, two genes encoding the RNA m6A methyltransferases, led to a decrease in the mRNA level of InR and underdevelopment of ovaries in B. dorsalis, and blocked the promoting effect of methionine on ovarian development and fecundity. Collectively, our study identifies an unrecognized role of RNA m6A methylation modification that underlies microbial control of host reproduction. Our findings further expand the functional landscape of m6A modification to include nutrient-dependent control of ovarian development and highlight the essential role of epigenetic regulation in microbe-host interactions.

microbiology↗

bioRxiv2024

Intestinal commensal bacteria promote Bactrocera dorsalis larval development through vitamin B6 synthesis pathway

BackgroundThe gut microbiota can facilitate host growth under nutrient-constrained conditions. However, whether this effect is limited to certain bacterial species remains largely unclear, and the relevant mechanisms remain to be thoroughly investigated. ResultsHere, we found that the microbiota was required for Bactrocera dorsalis larval growth under poor diet conditions. Mono-association experiments revealed that Enterobacteriaceae and some Lactobacilli promoted larval growth. Of 27 tested bacterial strains, 15 strains significantly promoted larval development, and the Enterobacteriaceae cloacae N29 isolate exhibited the most obvious promoting effect. Bacterial genome-wide association study (GWAS) revealed that the vitamin B6 synthesis pathway was critical for E. cloacae growth promotion. The deletion of pdxA responsible for the vitamin B6 biosynthesis deprived the mutant strains of larval growth promotion function, indicating that pdxA gene was crucial for promoting larval growth in the N29 strain. Importantly, supplementation of vitamin B6 to poor diet successfully rescued the axenic larval growth phenotype of B. dorsalis. ConclusionOur results suggest that gut microbes promote insect larval growth by providing vitamin B6 under nutrient scarcity in B. dorsalis.

microbiology↗

bioRxiv2023

Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow

The gut must perform a dual role of protecting the host against toxins and pathogens while harboring mutualistic microbiota. Previous studies suggested that the NADPH oxidase Duox contributes to intestinal homeostasis in Drosophila by producing reactive oxygen species (ROS) in the gut that stimulate epithelial renewal. We find instead that ROS generated by Duox in the Malpighian tubules leads to the production of Upd3, which enters the gut and stimulates stem cell proliferation. We describe for the first time in Drosophila the existence of a countercurrent flow system, which pushes tubule-derived Upd3 to the anterior part of the gut and stimulates epithelial renewal at a distance. Thus, our paper clarifies the role of Duox in gut homeostasis and describes the existence of retrograde fluid flow in the gut, collectively revealing a fascinating example of inter-organ communication.

immunology↗

bioRxiv2023

A humoral stress response protects Drosophila tissues from antimicrobial peptides

The immune response against an invading pathogen is generally associated with collateral tissue damage caused by the immune system itself. Consequently, several resilience mechanisms have evolved to attenuate the negative impacts of immune effectors. Antimicrobial peptides (AMPs) are small, cationic peptides that contribute to innate defenses by targeting negatively charged membranes of microbes1, 2. While being protective against pathogens, AMPs can be cytotoxic to host cells1, 3. Little is known of mechanisms that protect host tissues from AMP-induced immunopathology. Here, we reveal that a family of stress-induced proteins, the Turandots4, 5, protect Drosophila host tissues from AMPs, increasing resilience to stress. Deletion of several Turandot genes increases fly susceptibility to environmental stresses due to trachea apoptosis and poor oxygen supply. Tracheal cell membranes expose high levels of phosphatidylserine, a negatively charged phospholipid, sensitizing them to the action of AMPs. Turandots are secreted from the fat body upon stress and bind to tracheal cells to protect them against AMPs. In vitro, Turandot A binds to phosphatidylserine on membranes and inhibits the pore-forming activity of Drosophila and human AMPs on eukaryotic cells without affecting their microbicidal activity. Collectively, these data demonstrate that Turandot stress proteins mitigate AMP cytotoxicity to host tissues and therefore improve their efficacy. This provides a first example of a humoral mechanism used by animals limiting host-encoded AMP collateral damages.

immunology↗

bioRxiv2022

Single innate immune effectors control ecological microbiome bacteria across evolutionary timescales

Antimicrobial peptides are host-encoded immune effectors that combat pathogens and shape the microbiome in plants and animals. However, little is known about how the host antimicrobial peptide repertoire is adapted to its microbiome. Here we characterize the function and evolution of the Diptericin antimicrobial peptide family of Diptera. Using mutations affecting the two Diptericins (Dpt) of Drosophila melanogaster, we reveal the specific role of DptA for the pathogen Providencia rettgeri and DptB for the gut mutualist Acetobacter. Strikingly, presence of DptA- or DptB-like genes across Diptera correlates with the presence of Providencia and Acetobacter in their environment. Moreover, DptA- and DptB-like sequence predicts host resistance against infection by these bacteria across the genus Drosophila. Our study explains the evolutionary logic behind the bursts of rapid evolution of an antimicrobial peptide family, and reveals how the host immune repertoire adapts to changing microbial environments.

immunology↗

bioRxiv2022

Antimicrobial peptides do not directly contribute to aging in Drosophila, but improve lifespan by preventing dysbiosis

Antimicrobial peptides (AMPs) are innate immune effectors first studied for their role in host defense against bacterial and fungal infections. Recent studies have implicated these peptides in the clearance of aberrant cells and various neurological processes including neurodegenerative syndromes. In Drosophila, an array of AMPs are produced downstream of the Toll and Imd NF-{kappa}B pathways in response to infection. Many studies have suggested a role for the Imd pathway and AMPs in aging in this insect, supported by the upregulation of AMPs with aging (so-called "inflammaging"). However, functional studies using RNAi or over-expression have been inconclusive on whether and how these immune effectors impact aging. Leveraging a new set of single and compound AMP gene deletions in a controlled genetic background, we have investigated how AMPs contribute to aging. Overall, we found no major effect of individual AMPs on lifespan, with a possible exception of Defensin. However,{Delta} AMP14 flies lacking 14 AMP genes from seven families display a reduced lifespan. Interestingly, we found an increased bacterial load in the food medium of aged{Delta} AMP14 flies, suggesting that the lifespan reduction of these flies was due to a failure in controlling the microbiome. Consistent with this idea, use of germ-free conditions extends the lifespan of{Delta} AMP14 flies. Overall, our results do not point to an overt role of individual AMPs in lifespan. Instead, we find that AMPs collectively impact lifespan by preventing dysbiosis over aging. This is consistent with our previous study showing that AMPs control the gut microbiome, and many works showing that dysbiosis is detrimental upon aging. In the course of our experiments, we also uncovered a strong impact of a Drosophila nora virus infection on lifespan, and share our experience in reconciling our data given this confounding cryptic factor.

immunology↗

bioRxiv2022

Drosophila immunity: The Drosocin gene encodes two host defence peptides with pathogen-specific roles

1.1Antimicrobial peptides (AMPs) are key players in innate defence against infection in plants and animals. In Drosophila, many host defence peptides are produced downstream of the Toll and Imd NF-{kappa}B pathways. Use of single and compound AMP mutations in Drosophila has revealed that AMPs can additively or synergistically contribute to combat pathogens in vivo. However, these studies also revealed a high degree of specificity, wherein just one AMP can play a major role in combatting a specific pathogen. We recently uncovered a specific importance of the antibacterial peptide Drosocin for defence against Enterobacter cloacae. Here, we show that the Drosocin locus (CG10816) is more complex than previously described. In addition to its namesake peptide "Drosocin", it encodes a second peptide generated from a precursor via furin cleavage. We name this peptide "Buletin", and show that it corresponds to the uncharacterized "Immune-induced Molecule 7" previously identified by MALDI-TOF. The existence of a naturally occurring polymorphism (Thr52Ala) in the CG10816 precursor protein masked the identification of this peptide previously. Using mutations differently affecting the production of these two CG10816 gene products, we show that Drosocin, but not Buletin, contributes to the CG10816-mediated defence against E. cloacae. Strikingly, we observed that Buletin, but not Drosocin, contributes to the CG10816-mediated defence against Providencia burhodogranariea. Moreover, the Thr52Ala polymorphism in Buletin affects survival to P. burhodogranariea, wherein the Alanine allele confers better defence than the Threonine allele. However, we found no activity of Buletin against either P. burhodogranariea or E. coli in vitro. Collectively, our study reveals that CG10816 encodes not one but two prominent host defence peptides with different specificity against different pathogens. This finding emphasizes the complexity of the Drosophila humoral response consisting of multiple host defence peptides with specific activities, and demonstrates how natural polymorphisms found in Drosophila populations can affect host susceptibility.

immunology↗

bioRxiv2021

The wall-less bacterium Spiroplasma poulsonii builds a polymeric cytoskeleton composed of interacting MreB isoforms

A rigid cell wall defines the morphology of most bacteria. MreB, a bacterial homologue of actin, plays a major role in coordinating cell wall biogenesis and defining a cells shape. In contrast with most bacteria, the Mollicutes family is devoid of cell wall. As a consequence, many Mollicutes have undefined morphologies. Spiroplasma species are an exception as they robustly grow with a characteristic helical shape, but how they maintain their morphology remains unclear. Paradoxal to their lack of cell wall, the genome of Spiroplasma contains five homologues of MreB (SpMreBs). Since MreB is a homolog of actin and that short MreB filaments participate in its function, we hypothesize that SpMreBs form a polymeric cytoskeleton. Here, we investigate the function of SpMreB in forming a polymeric cytoskeleton by focusing on the Drosophila endosymbiont Spiroplasma poulsonii. We found that in vivo, Spiroplasma maintain a high concentration of all five MreB isoforms. By leveraging a heterologous expression system that bypasses the poor genetic tractability of Spiroplasma, we found that strong intracellular levels of SpMreb systematically produced polymeric filaments of various morphologies. Using co-immunoprecipitation and co-expression of fluorescent fusions, we characterized an interaction network between isoforms that regulate the filaments formation. Our results point to a sub-functionalization of each isoform which, when all combined in vivo, form a complex inner polymeric network that shapes the cell in a wall-independent manner. Our work therefore supports the hypothesis where MreB mechanically supports the cell membrane, thus forming a cytoskeleton. Significance statementBacteria shape is determined by their cell wall. The actin homologue MreB essentially determines shape by organizing cell wall synthesis at the subcellular level. Despite their lack of cell wall, Spiroplasma robustly grow into long helical bacteria. Surprisingly, its genome retains five copies of mreB while it lost genes encoding canonical MreB interactors. We sought to delineate the exact function of Spiroplasma MreBs (SpMreBs). We leveraged in vivo data along with functional studies to systematically investigate MreB polymerization behavior. We uncovered that SpMreBs build into filaments, which structure it determined by a complex interaction network between isoforms. Our results support the hypothesis that MreB can mechanically support the membrane of Spiroplasma, hence acting as a load-bearing cytoskeletal protein.

microbiology↗