Biology subjects

Cascales, E.

Publications and source records attributed to Cascales, E..

11 recordsLinked to original sources

bioRxiv2026

Insights Into an Antibacterial Type VI Membrane-Disrupting Effector Of Adherent Invasive Escherichia coli

Adherent Invasive Escherichia coli (AIEC) strains are associated with ileal Crohn's disease and may contribute to intestinal dysbiosis through antibacterial competition. The AIEC reference strain LF82 encodes two type VI secretion systems (T6SSs), a contractile machine that injects an effector-loaded needle made of Hcp, VgrG and PAAR into neighbouring rivals, favoring niche colonization. LF82 T6SSs harbour several putative antibacterial toxin-immunity modules. Here we characterize the T6SS-1 encoded TmeAIEC, a PAAR-fused T6SS effector carrying a C-terminal Tme domain, hallmark of Tme (Type VI membrane disrupting effector) toxins, and its DUF1240-like immunity protein TmiAIEC. When heterologously produced in E. coli K-12 as well as in E. coli and Citrobacter clinical isolates, TmeAIEC is toxic only upon periplasmic targeting, and its toxicity is neutralized by TmiAIEC. Periplasmically-targeted TmeAIEC inserts into the inner membrane, collapses the proton motive force and increases cell permeability, consistent with a membrane-disrupting activity. Mutational analyses coupled to functional assays identified a predicted hydrophobic transmembrane segment and a conserved aspartate residue within the Tme domain as key determinants for TmeAIEC toxicity. These findings establish TmeAIEC/TmiAIEC as a functional T6SS toxin-immunity pair and reveal conserved determinants underlying the activity of Tme-family membrane-disrupting effectors.

microbiology↗

bioRxiv2026

Aggregation-mediated microcolony shields bacteria from contact-dependent competition and maintains phenotypic heterogeneity

Bacteria are adaptable microorganisms capable of surviving in a wide variety of environments. One common strategy for persistence and attachment to surfaces is the formation of microcolonies, often controlled by cell surfaces structures that mediate adhesion such as fimbriae. In this study, we show that type 1 fimbriae, and other adhesins structures such as autotransporter adhesins, trigger microcolony formation which confer resistance against a broad range of short-range contact-dependent weapons (e.g. T6SS, T4SS, CDI) but not against long-range diffusible weapons (e.g. diffusible toxins like colicins). Interestingly, we identify "sheltered" bacteria within microcolonies that benefit from collective protection without expressing the adhesive structure. Our findings demonstrate that adhesive structures not only improve survival in hostile environments by promoting microcolony formation but also maintain phenotypic heterogeneity within the microcolony, highlighting the importance of social behavior in bacterial adaptation to changing environments.

microbiology↗

bioRxiv2026

An ART-fold Rhs toxin from Pluralibacter gergoviae defines Tne5, a new clade of NAD(P)⁺ glycohydrolases effectors

The type VI secretion system (T6SS) ia a widespread bacterial nanomachine that mediates interbacterial competition by delivering toxic effectors into neighboring cells. Among these, enzymes targeting nicotinamide adenine dinucleotide cofactors (NAD and NADP) are particularly potent because they rapidly disrupt redox homeostasis and central metabolism. Several families of T6SS-associated NAD(P)-consuming effectors (Tne1-Tne4) have been described. Here, we characterize a T6SS-associated Rhs toxin from Pluralibacter gergoviae. Competition assays show that P. gergoviae kills Escherichia coli in a T6SS-dependent manner. Heterologous production reveals that the Rhs C-terminal extension is toxic in the E. coli cytoplasm and that co-production with the protein encoded downstream neutralizes this activity. AlphaFold3 modeling predicts that the toxin adopts an ADP-ribosyltransferase (ART)-like /{beta} fold with a putative catalytic pocket accommodating NAD. By contrast to T6SS ART toxins described so far, the toxin does not inhibit transcription, translation or cell division, but instead depletes NAD and NADP. Phylogenetic analyses and structural modeling show that this effector defines a new ART-related family of NAD(P) glycohydrolases, which we propose to name Tne5, broadly distributed across antagonistic systems.

microbiology↗

bioRxiv2025

Widespread repurposing of the human CD38 ADP-ribosyl cyclase fold for toxicity in antibacterial and anti-eukaryotic bacterial polymorphic toxins

Bacterial polymorphic toxins are modular weapons that mediate inter-microbial competition and host interactions by delivering diverse cytotoxic domains through specialized secretion systems. Here, we identify and characterize a novel toxin domain in Pantoea ananatis that displays remarkable structural and functional conservation with the human enzyme CD38. This bacterial toxin, fused to the type VI secretion system (T6SS) PAAR domain, harbors a C-terminal ADP-ribosyl cyclase (ARC) domain that hydrolyzes NAD+ and NADP+ in vitro and in vivo, leading to growth inhibition in both bacterial and eukaryotic cells. The 1.6-[A] resolution structure of ARC reveals that it adopts a globular fold nearly identical to the human CD38 ADP ribosyl cyclase, with key catalytic residues conserved. ARC toxicity is neutralized in P. ananatis by a dedicated immunity protein. Comparative genomics reveals that CD38-like ARC domains are widespread in bacteria, fused to diverse delivery modules including T6SS, T7SS, and CDI systems. Functional assays demonstrate that these domains act as NAD-depleting toxins, with cross-immunity observed between non-cognate toxin-immunity pairs. Taken together, our findings reveal that a eukaryotic-like NAD+ hydrolase fold has been adapted in bacteria to generate a novel class of metabolic toxins, expanding the functional scope of polymorphic effectors and illustrating how conserved host-like enzymes can be co-opted for microbial warfare.

microbiology↗

bioRxiv2025

Type VI secretion phenotypic heterogeneity ensures trade-off between antibacterial activity and resistance in Enteroaggregative E. coli

The type VI secretion system (T6SS) is a nanoweapon deployed by bacteria to deliver effectors into target cells, allowing niche colonization or host invasion. In entero-aggregative Escherichia coli (EAEC), the expression of the T6SS gene cluster is responsive to iron starvation by the Fur regulator, and to Dam-dependent methylation. However, fluorescence microscopy recordings showed that the T6SS displays heterogeneous expression in a clonal population. The population is composed of three types of cells in a stable equilibrium: non producers (OFF), producers, and assemblers (ON). Single-cell analyses further showed that the activation state is transmitted to the progeny, but the overall distribution respects the equilibrium suggesting a genetic control of heterogeneity. Mutagenesis of Fur-binding boxes and GATC sites revealed that specific promoter elements control this heterogeneity. Phenotypic characterization of the ON and OFF subpopulations demonstrated that ON cells eliminate susceptible target cells, whereas OFF cells survive attacks from T6SS-defensive bacteria. The heterogeneity of EAEC T6SS is therefore an attenuation strategy conferring a trade-off between antibacterial efficiency and survival in polymicrobial environments.

microbiology↗

bioRxiv2025

Molecular bases for the loss of type VI secretion system activity during enteroaggregative E. coli experimental evolution

The type VI secretion system (T6SS) is a nanoweapon deployed by Gram-negative to inject effectors into target cells and hence involved in pathogenesis and bacterial competition. While T6SS gene clusters are found in all recently isolated commensals or pathogenic Escherichia coli strains, they are absent from classical laboratory strains. These E. coli strains, which have been used since decades for bacterial genetics, were usually grown in pure cultures, suggesting that T6SS might have been lost during evolution in absence of competitors. Here, we conducted a 640-generation experimental evolution by passaging the enteroaggregative E. coli (EAEC) 17-2 strain under controlled competition conditions against susceptible or immune recipient cells. EAEC T6SS activity was almost abolished when grown in the presence of immune recipients, while no difference with the ancestral strain was observed for EAEC grown in the presence of susceptible cells. Whole genome sequencing of 18 clones identified adaptative mutations responsible for the loss of T6SS activity, including mutations of the T6SS promoter and within genes encoding T6SS subunits, and revealed a novel regulatory mechanism involving RfaH-dependent antitermination. We further found that the RfaH binding site ops element is present in T6SS gene clusters of several species, suggesting a conserved RfaH-dependent regulation of T6SSs across enterobacteria. This work exemplifies the power of experimental evolution to understand T6SS genetic adaptation and to unravel new players for its function.

microbiology↗

bioRxiv2021

Dynamic proton-dependent motors power Type IX secretion and gliding adhesin movement in Flavobacterium

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMH) and show that conserved glutamate residues in GldL TMH are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

microbiology↗

bioRxiv2021

A Tad-like apparatus is required for contact-dependent prey killing in predatory social bacteria

Myxococcus xanthus, a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contac with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages.

microbiology↗

bioRxiv2020

Central role and structure of the membrane pseudokinase YukC in the antibacterial Bacillus subtilis Type VIIb Secretion System.

Type VIIb Secretion System (T7SSb) has been recently identified in Firmicutes resembling the mycobacterial T7SSa. Despite limited sequence homology, T7SSa and T7SSb have substrates with striking structural similarities, the WXG100 proteins. Recent advances in Staphylococcus spp. proposed that T7SSb is involved in intra-species competition. However, the architecture and mechanism of action of this secretion complex remain largely obscure. Here, we investigate the T7SSb of Bacillus subtilis as a model system. We report the first evidence of B. subtilis ability to mediate intra- and inter-species antibacterial activity in a T7SSb-dependent manner. Then, we present the first systematic investigation of the T7SSb protein-protein network, revealing novel interactions and highlighting the central role of the pseudokinase subunit YukC in the assembly of the system. Its direct interaction with a T7SSb-secreted toxin supports its role in recruiting substrates to the secretion machinery. Finally, we solved the crystal structure of full-length transmembrane YukC defining novel structural motifs and suggesting that intrinsic flexibility modulates the orientation of the pseudokinase domains and YukC function. Overall, our results provide a better understanding on the role and molecular organisation of the T7SSb, opening new perspectives for the comprehension of this poorly characterized molecular machine.

microbiology↗

bioRxiv2018

A conserved motif of Porphyromonas Type IX secretion effectors C-terminal secretion signal specifies interactions with the PorKLMN core complex

The Type IX secretion system (T9SS) is a versatile protein transport apparatus restricted to the Bacteroidetes phylum. This multiprotein complex enables secretion of a wide range of effectors, such as protein toxins, filamentous adhesins, enzymes or S-layer subunits. Once translocated in the periplasm through the Sec pathway, recognition and secretion of these cargo proteins rely on a conserved C-terminal domain referred as CTD. However, the precise route followed by the CTD substrates from the periplasm to the cell exterior is yet to be determined. Here we define the interaction network of five different CTDs from the oral pathogen Porphyromonas gingivalis with the PorKLMN T9SS trans-envelope complex. We show that these five CTDs interact with the inner membrane-anchored PorM and the PorN periplasmic proteins. We further determine the contribution of the PorM IgG-like periplasmic domains and CTD conserved motifs for PorM-CTD complex formation. These results showed that all five CTDs interact with the core complex in a similar manner, suggesting a conserved mechanism of substrate selection in the periplasm. Our results thus establish a first model of the path followed by the CTD substrates through the T9SS.

microbiology↗

bioRxiv2018

In situ and high-resolution Cryo-EM structure of the Type VI secretion membrane complex

Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. The T6SS is composed of a baseplate that contains a spike onto which an inner tube is built, surrounded by a contractile sheath. Unlike phages that are released to and act in the extracellular medium, the T6SS is an intracellular machine inserted in the bacterial membranes by a trans-envelope complex. This membrane complex (MC) comprises three proteins: TssJ, TssL and TssM. We previously reported the low-resolution negative stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tomography analysis of the T6SS MC confirmed the 5-fold symmetry in situ and identified the regions of the structure that insert into the bacterial membranes. A high resolution model obtained by single particle cryo-electron microscopy reveals its global architecture and highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, a 11-residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we revisit the model on the mechanism of action of the MC during T6SS assembly and function.

microbiology↗