Biology subjects

Chenal, A.

Publications and source records attributed to Chenal, A..

6 recordsLinked to original sources

bioRxiv2026

A Seven-Protein Assembly Promotes Stability, Neutralisation and Secretion of the T7SSb LXG-effector TelE

Streptococcus gallolyticus subsp. gallolyticus (SGG) is a gut pathobiont associated with colorectal cancer. Like many Firmicutes, SGG utilizes a specialized Type VII Secretion System (T7SSb) to export WXG100 and LXG proteins, the latter involved in bacterial competition. We previously identified TelE, an LXG protein whose C-terminus mediates membrane pore formation in Escherichia coli. In SGG UCN34, TelE (Gallo_0562) is co-expressed with six proteins (Gallo_0559 to Gallo_0565), including its immunity protein TipE (Gallo_0565). Here we show that the absence of those co-expressed proteins affects TelE stability and secretion. These proteins associate with TelE to form a soluble and stable seven-protein complex. Gallo_0559 and Gallo_0560 interact with the N-terminal LXG domain of TelE, Gallo_0561 binds to its central region, while the six-transmembrane protein Gallo_0563, together with Gallo_0564 and TipE associates with its C-terminal domain. These findings describe a new modular complex that stabilizes TelE while reducing its toxicity and optimizing its T7SSb-mediated delivery.

microbiology↗

bioRxiv2023

Genome sequences of equine influenza A subtype H3N8 viruses by long read sequencing and functional characterization of the PB1-F2 virulence factor of A/equine/Paris/1/2018

Equine influenza virus (EIV) remains a persistent threat to equines, despite the availability of vaccines. Currently, strategies to monitor the virus and prevent any potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for swift and precise sequencing of circulating equine influenza viruses. To this end, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently used OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were located in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential {Delta}{Psi}m and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

microbiology↗

bioRxiv2022

Gallocin A, an atypical two-peptide bacteriocin secreted by Streptococcus gallolyticus subsp. gallolyticus

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed at characterizing the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives. Importantly, we demonstrated that gallocin A peptides can insert into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon named GIP, is necessary and sufficient to confer immunity to gallocin A. Structural modelling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired gallocin A ability to kill Enterococcus faecalis. Structural modelling of GIP revealed a hairpin-like structure strongly resembling that of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and often associated with asymptomatic colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide to SGG with the ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (Aymeric et al., 2017). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin and renamed gallocin A recently following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and to kill phylogenetically closely related bacteria such as most streptococci, lactococci and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and with very few charged amino acids compared to well-known class IIb bacteriocins. In silico modelling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide also forms a hairpin like structure like GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin displaying disulfide bridges important for its structure and activity and the founding member of a subtype of class IIb bacteriocins.

microbiology↗

bioRxiv2020

A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

biophysics↗

bioRxiv2020

Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at the low concentrations that may be found in vivo during B. pertussis infection, CyaA impairs the migration and wound healing capacities of the intoxicated alveolar epithelial cells. Our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, that is an hallmark of pertussis.

cell biology↗

bioRxiv2019

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, one of the largest bacterial phyla that includes the deadly human pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors as well as all positive and negative Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to directly interact with the conserved C-terminal tail of FtsZ but whose actual mode of action in cytokinesis is yet to be elucidated. We used a mechanistic cell biology approach to unveil the essential interdependence of FtsZ and SepF required for the formation of a functional Z-ring in the actinobacterial model organism C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and a reversible oligomerization interface regulated via an alpha helical switch. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, leading to a complex dynamic role of the protein at the division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

microbiology↗