Biology subjects

Filloux, A.

Publications and source records attributed to Filloux, A..

10 recordsLinked to original sources

bioRxiv2025

PI3Kδ Bridges Microbial Surveillance with Antigen Presentation to Reinforce Intestinal Immunity

Phosphoinositide 3-kinase delta (PI3K{delta}) is essential for immune cell functions, preventing immunodeficiency and inflammation; however, its role in dendritic cell (DC)-mediated immune regulation remains unknown. Here, we report a key role for DC-intrinsic PI3K{delta} in linking microbial recognition with antigen presentation for effective T-cell priming. Using genetic and functional assays, we demonstrate that PI3K{delta} deficiency in DCs leads to broad dysregulation of intestinal CD4 T-cell immunity, characterized by impaired regulatory T-cell expansion and increased susceptibility to colitis. DC-based studies show that PI3K{delta} links pattern-recognition-receptor signaling to MHC class I- and II-restricted presentation of phagosome-associated antigens by facilitating NOX2-dependent oxidative burst through RAC2, while mitigating inflammasome activation. In contrast, PI3K{delta} deficiency disrupts phagosomal pH balance, leading to accelerated acidification and proteolysis of antigens, impairing T-cell activation. Our study identifies PI3K{delta} as a key coordinator of phagosome dynamics, important for DC adaptive programming that shapes T-cell responses and supports intestinal immune homeostasis.

immunology↗

bioRxiv2023

Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis

Critical Gram-negative pathogens, like Pseudomonas, Stenotrophomonas and Burkholderia, have become resistant to most antibiotics. Complex resistance profiles together with synergistic interactions between these organisms increase the likelihood of treatment failure in distinct infection settings, for example in the lungs of cystic fibrosis (CF) patients. Here, we discover that cell envelope protein homeostasis pathways underpin both antibiotic resistance and cross-protection in CF-associated bacteria. We find that inhibition of oxidative protein folding inactivates multiple species-specific resistance proteins. Using this strategy, we sensitize multidrug-resistant Pseudomonas aeruginosa to {beta}-lactam antibiotics and demonstrate promise of new treatment avenues for the recalcitrant emerging pathogen Stenotrophomonas maltophilia. The same approach also inhibits cross-protection between resistant S. maltophilia and susceptible P. aeruginosa, allowing eradication of both commonly co-occurring CF-associated organisms. Our results provide the basis for the development of next-generation strategies that target antibiotic resistance, while also impairing specific interbacterial interactions that enhance the severity of polymicrobial infections.

microbiology↗

bioRxiv2022

Effectiveness of Pseudomonas aeruginosa type VI secretion system relies on toxin potency and type IV pili-dependent interaction

The type VI secretion system (T6SS) is an antibacterial weapon that is used by numerous Gram-negative bacteria to gain competitive advantage by injecting toxins into adjacent prey cells. Predicting the outcome of a T6SS-dependent competition is not only reliant on presence-absence of the system but instead involves a multiplicity of factors. Pseudomonas aeruginosa possesses 3 distinct T6SSs and a set of more than 20 toxic effectors with diverse functions including disruption of cell wall integrity, degradation of nucleic acids or metabolic impairment. We generated a comprehensive collection of mutants with various degrees of T6SS activity and/or sensitivity to each individual T6SS toxin. By imaging whole mixed bacterial macrocolonies, we then investigated how these P. aeruginosa strains gain a competitive edge in multiple attacker/prey combinations. We observed that the potency of single T6SS toxin varies significantly from one another as measured by monitoring the community structure, with some toxins acting better in synergy or requiring a higher payload. Remarkably the degree of intermixing between preys and attackers is also key to the competition outcome and is driven by the frequency of contact as well as the ability of the prey to move away from the attacker using type IV pili-dependent twitching motility. Finally, we implemented a computational model to better understand how changes in T6SS firing behaviours or cell-cell contacts lead to population level competitive advantages, thus providing conceptual insight applicable to all types of contact-based competition.

microbiology↗

bioRxiv2022

Characterization of TelD, an LXG effector of Streptococcus gallolyticus, antagonized by a non-canonical immunity protein

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the Type VIIb Secretion System (T7SSb) machinery is uniquely present in SGG in two different arrangements. SGG UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by this secretion system, we inactivated the essC gene encoding the motor of this machinery. Comparison of the proteins secreted by UCN34 WT and its isogenic {Delta}essC mutant revealed six T7SSb effector proteins, including the expected WXG effector EsxA and three LXG-containing proteins. In this work, we characterized an LXG-family toxin named herein TelE displaying pore-forming activity. Seven homologs of TelE harboring a conserved glycine zipper motif at the C-terminus were identified in different SGG isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelE pore-forming activity. Unlike other pore-forming toxins commonly antagonized by a membrane protein, TelE activity was antagonized by a small protein TipE belonging to the DUF5085 family. Overall, we report herein a unique SGG T7SSb effector exhibiting a pore-forming activity against non-immune bacteria. IMPORTANCEIn this study, 38 clinical isolates of Streptococcus gallolyticus subsp. gallolyticus (SGG) were sequenced and a genetic locus encoding the Type VIIb secretion system (T7SSb) was found conserved and absent from 16 genomes of the closely related S. gallolyticus subsp. pasteurianus (SGP). The T7SSb is a bona fide pathogenicity island. Here, we report that the model organism SGG strain UCN34 secretes six T7SSb effectors. One of the six effectors named TelE displayed a strong toxicity when overexpressed in Escherichia coli. Our results indicate that TelE is a pore forming toxin whose activity can be antagonized by a non-canonical immunity protein named TipE. Overall, we report a unique toxin-immunity protein pair and our data expand the range of effectors secreted through T7SSb.

microbiology↗

bioRxiv2022

Architecture of the biofilm-associated archaic CupE pilus from Pseudomonas aeruginosa

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE pili consist of CupE1 subunits arranged in a zigzag architecture, with an N-terminal donor {beta}-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, resulting in an overall flexible pilus arrangement. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which may facilitate their role in promoting cohesion between bacterial cells. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili and their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.

microbiology↗

bioRxiv2021

Structure of ATP synthase from ESKAPE pathogen Acinetobacter baumannii

Acinetobacter baumannii is a clinically relevant pathogen which causes multi-drug resistant, hospital-acquired infections and is a top priority target for antibiotic development. Cryo-EM structures of the A. baumannii F1Fo-ATP synthase in three conformational states reveal unique features, which represent attractive sites for the development of novel therapeutics. One sentence summaryStructure of Acinetobacter baumannii ATP synthase

biochemistry↗

bioRxiv2020

Novel structural components generate distinct type VI secretion system anchoring modes

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short (TssAS) and a long (TssAL) TssA. Whilst TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a novel class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for a while or fire immediately after sheath extension, thus giving rise to different aggression behaviors. We propose that this functional diversity could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

microbiology↗

bioRxiv2019

Fast and robust detection of colistin resistance in Escherichia coli using the MALDI Biotyper Sirius mass spectrometry system

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by MALDI-TOF mass spectrometry in less than 15 minutes but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF mass spectrometry system (Bruker Daltonics). We optimized the sample preparation protocol using a set of 6 MCR-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 MCR producers, 12 chromosomally-resistant isolates and 9 polymyxin-susceptible isolates. We calculated Polymyxin resistance ratio (PRR) values from the acquired spectra; a PRR value of zero, indicating polymyxin susceptibility, was obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test, adapted for the routine MALDI Biotyper Sirius, provides an unbiased, fast, reliable, cost-effective and high-throughput way of detecting colistin resistance in clinical E. coli isolates.

microbiology↗

bioRxiv2019

Discovery of a Pseudomonas aeruginosa Type VI secretion system toxin targeting bacterial protein synthesis using a global genomics approach

The Type VI secretion system (T6SS) is a bacterial weapon which delivers toxic effectors to kill competitors or subvert some of their key functions. Here we use transposon directed insertion-site sequencing (TraDIS) to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria lacking either one or both of the asparagine or glutamine tRNA synthases. Our data suggests that Tse8 combines as a non-cognate component of the transamidosome complex, reducing fitness by limiting the ability of the cell to synthesize proteins. This is the first demonstration of a T6SS toxin affecting protein synthesis, expanding the range of cellular components targeted by this bacterial weapon. The success of the current study validates the use of our TraDIS approach as a tool to drastically expand the repertoire of T6SS toxins in any T6SS-encoding bacterium.

microbiology↗

bioRxiv2018

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggests a differential effect on twitching motility by flagellum components based upon their cellular location. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

genomics↗