Biology subjects

Francetic, O.

Publications and source records attributed to Francetic, O..

5 recordsLinked to original sources

bioRxiv2026

Structural determinants of endopilus assembly, stability and functional specificity in bacterial type II secretion

Gram-negative bacteria employ the type II secretion system (T2SS) to transport folded protein effectors across the outer membrane. This multiprotein nanomachine assembles the endopilus, a periplasmic helical polymer composed of one major and four minor pilin subunits. Endopili are structurally related to type IV pili but exhibit distinct features including a conserved calcium-binding site stabilizing their major pilin subunits. Endopilus polymerisation is coupled to substrate translocation through a dedicated outer membrane channel. To investigate the structural basis of endopilus assembly, stability and functional specificity, we performed a comparative analysis of the Out T2SS from the plant pathogen Dickeya dadantii and the Pul T2SS from the human pathogen Klebsiella oxytoca. Although their major pilins OutG and PulG share over 77% sequence identity, these bacteria differ markedly in ecological niche and range of secreted effectors. We report here the NMR structure of calcium-bound OutG monomer and cryo-EM structures of OutG and PulG endopili at 3.6 [A] resolution. The integration of structural, mutational, and biophysical analyses with in vivo assays, allowed us to identify the molecular determinants of secretion specificity and endopilus stability. These findings demonstrate how subtle sequence variations in conserved nanomachines have evolved to optimize their function and adapt to their local environments.

microbiology↗

bioRxiv2022

Periplasmic coiled coil formed by assembly platform proteins PulL and PulM is critical for function of the Klebsiella type II secretion system

Bacteria use type II secretion systems (T2SS) to secrete to their surface folded proteins that confer diverse functions, from nutrient acquisition to virulence. In the Klebsiella species, T2SS-mediated secretion of pullulanase (PulA) requires assembly of a dynamic filament called pseudopilus. The inner membrane assembly platform (AP) complex is essential for PulA secretion and pseudopilus assembly. The AP components PulL and PulM form an inner membrane complex interacting through their C-terminal globular domains and transmembrane segments. Here we investigated the roles of periplasmic helices and cytoplasmic domains of PulL and PulM in their assembly. We found that PulL and PulM variants lacking periplasmic helices were defective for interactions in the bacterial two-hybrid (BACTH) assay. Their function in PulA secretion and assembly of PulG subunits into pseudopilus filaments were strongly reduced. In addition, deleting the cytoplasmic peptide of PulM in variant PulM{Delta}N nearly abolished interaction with PulG in the BACTH assay, without affecting the interaction with PulL. Nevertheless, PulL was degraded in the presence of the PulM{Delta}N variant, suggesting that PulM N-terminal peptide interacts with PulL in the cytoplasm and plays a stabilizing role. We discuss the implication of these results for the mechanism of T2S and type IV pilus assembly.

molecular biology↗

bioRxiv2022

Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system

Type II secretion systems (T2SS) allow diderm bacteria to secrete hydrolytic enzymes, adhesins or toxins important for growth and virulence. In T2SS, secretion of folded proteins from the periplasm to the cell surface requires assembly of periplasmic filaments called pseudopili. Like the related type IV pili, pseudopili are polymerized in the inner membrane through addition of subunits at the filament base, mediated by the essential assembly platform (AP). To understand the structure and molecular role of the AP, we focused on its components PulL and PulM from the Klebsiella oxytoca T2SS. By combining biophysical methods, NMR and X-ray crystallography we studied the structure and associations of their periplasmic domains. We describe the first structure of the heterodimer complex formed by the PulL and PulM ferredoxin-like domains and show how their structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and cross-linking of transmembrane segments provided additional constraints to build a structural model of the PulL-PulM complex and assembly in the cellular context. Together with the relative abundance of PulL, PulM and their partners our findings suggest a model of the AP as a dynamic hub that orchestrates pseudopilus polymerization.

microbiology↗

bioRxiv2021

Computational and biochemical analysis of type IV Pilus dynamics and stability

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating Nuclear Magnetic Resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability and function, we performed a total of 108 s all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH and ionic strength was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

biochemistry↗

bioRxiv2019

Ancestral Mitochondrial Protein Secretion Machinery

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.

evolutionary biology↗