BotCl, the First Chlorotoxin-like Peptide Inhibiting Newcastle Disease Virus: The Emergence of a New Scorpion Venom AMPs Family.

Newcastle disease virus (NDV) is one of the most serious contagions affecting domestic poultry and other avian species. It causes high morbidity and mortality, resulting in huge economic losses to the poultry industry worldwide. Despite vaccination, NDV outbreaks increase the need for alternative prevention and control means. In this study, we have screened fractions of Buthus occitanus tunetanus (Bot) scorpion venom and isolated the first scorpion peptide inhibiting the NDV multiplication. It showed a dose dependent effect on NDV growth in vitro, with an IC50 of 0.69 µM, and a low cytotoxicity on cultured Vero cells (CC50 > 55 µM). Furthermore, tests carried out in specific pathogen-free embryonated chicken eggs demonstrated that the isolated peptide has a protective effect on chicken embryos against NDV, and reduced by 73% the virus titer in allantoic fluid. The N-terminal sequence, as well as the number of cysteine residues of the isolated peptide, showed that it belongs to the scorpion venom Chlorotoxin-like peptides family, which led us to designate it "BotCl". Interestingly, at 10 µg/mL, BotCl showed an inhibiting effect three times higher than its analogue AaCtx, from Androctonus australis (Aa) scorpion venom, on NDV development. Altogether, our results highlight the chlorotoxin-like peptides as a new scorpion venom AMPs family.

Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.

Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.

Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains.

BACKGROUND: In a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity. RESULTS: We introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins. CONCLUSIONS: We show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Expansion of nickel binding- and histidine-rich proteins during gastric adaptation of Helicobacter species.

Acquisition and homeostasis of essential metals during host colonization by bacterial pathogens rely on metal uptake, trafficking, and storage proteins. How these factors have evolved within bacterial pathogens is poorly defined. Urease, a nickel enzyme, is essential for Helicobacter pylori to colonize the acidic stomach. Our previous data suggest that acquisition of nickel transporters and a histidine-rich protein (HRP) involved in nickel storage in H. pylori and gastric Helicobacter spp. have been essential evolutionary events for gastric colonization. Using bioinformatics, proteomics, and phylogenetics, we extended this analysis to determine how evolution has framed the repertoire of HRPs among 39 Epsilonproteobacteria; 18 gastric and 11 non-gastric enterohepatic (EH) Helicobacter spp., as well as 10 other Epsilonproteobacteria. We identified a total of 213 HRPs distributed in 22 protein families named orthologous groups (OGs) with His-rich domains, including 15 newly described OGs. Gastric Helicobacter spp. are enriched in HRPs (7.7 ± 1.9 HRPs/strain) as compared to EH Helicobacter spp. (1.9 ± 1.0 HRPs/strain) with a particular prevalence of HRPs with C-terminal histidine-rich domains in gastric species. The expression and nickel-binding capacity of several HRPs was validated in five gastric Helicobacter spp. We established the evolutionary history of new HRP families, such as the periplasmic HP0721-like proteins and the HugZ-type heme oxygenases. The expansion of histidine-rich extensions in gastric Helicobacter spp. proteins is intriguing but can tentatively be associated with the presence of the urease nickel enzyme. We conclude that this HRP expansion is associated with unique properties of organisms that rely on large intracellular nickel amounts for their survival.

Design and in vitro characterization of RXR variants as tools to investigate the biological role of endogenous rexinoids.

Retinoid X receptors (RXRα, ß, and γ) are essential members of the nuclear receptor (NR) superfamily of ligand-dependent transcriptional regulators that bind DNA response elements and control the expression of large gene networks. As obligate heterodimerization partners of many NRs, RXRs are involved in a variety of pathophysiological processes. However, despite this central role in NR signaling, there is still no consensus regarding the precise biological functions of RXRs and the putative role of the endogenous ligands (rexinoids) previously proposed for these receptors. Based on available crystal structures, we introduced a series of amino acid substitutions into the ligand-binding pocket of all three RXR subtypes in order to alter their binding properties. Subsequent characterization using a battery of cell-based and in vitro assays led to the identification of a double mutation abolishing the binding of any ligand while keeping the other receptor functions intact and a triple mutation that selectively impairs interaction with natural rexinoids but not with some synthetic ligands. We also report crystal structures that help understand the specific ligand-binding capabilities of both variants. These RXR variants, either fully disabled for ligand binding or retaining the property of being activated by synthetic compounds, represent unique tools that could be used in future studies to probe the presence of active endogenous rexinoids in tissues/organs and to investigate their role in vivo. Last, we provide data suggesting a possible involvement of fatty acids in the weak interaction of RXRs with corepressors.

Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog.

ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB's are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.

A ruthenium-oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy.

Ruthenium complexes have emerged as a promising class of compounds for use as photosensitizers (PSs) in photodynamic therapy (PDT) due to their attractive photophysical properties and relative ease of chemical alteration. While promising, they generally are not inherently targeting to disease sites and may therefore be prone to side effects and require higher doses. Aptamers are short oligonucleotides that bind specific targets with high affinity. One such aptamer is AS1411, a nucleolin targeting, G-quadruplex forming, DNA aptamer. Here we present the first example of direct conjugation of a Ru(ii) polypyridyl complex-based PS to an aptamer and an assessment of its in vitro cancer cell specific photosensitization including discussion of the challenges faced.

De Novo Sequencing of Antibody Light Chain Proteoforms from Patients with Multiple Myeloma.

In multiple myeloma diseases, monoclonal immunoglobulin light chains (LCs) are abundantly produced, with, as a consequence in some cases, the formation of deposits affecting various organs, such as the kidney, while in other cases remaining soluble up to concentrations of several g·L-1 in plasma. The exact factors crucial for the solubility of LCs are poorly understood, but it can be hypothesized that their amino acid sequence plays an important role. Determining the precise sequences of patient-derived LCs is therefore highly desirable. We establish here a novel de novo sequencing workflow for patient-derived LCs, based on the combination of bottom-up and top-down proteomics without database search. PEAKS is used for the de novo sequencing of peptides that are further assembled into full length LC sequences using ALPS. Top-down proteomics provides the molecular masses of proteoforms and allows the exact determination of the amino acid sequence including all posttranslational modifications. This pipeline is then used for the complete de novo sequencing of LCs extracted from the urine of 10 patients with multiple myeloma. We show that for the bottom-up part, digestions with trypsin and Nepenthes digestive fluid are sufficient to produce overlapping peptides able to generate the best sequence candidates. Top-down proteomics is absolutely required to achieve 100% final sequence coverage and characterize clinical samples containing several LCs. Our work highlights an unexpected range of modifications.

Mass spectrometry-based top-down and bottom-up approaches for proteomic analysis of the Moroccan Buthus occitanus scorpion venom.

Buthus occitanus (B. occitanus) is one of the most dangerous scorpions in the world. Despite the involvement of B. occitanus scorpion in severe cases of envenomation in Morocco, no study has focused yet on the proteomic composition of the Moroccan B. occitanus scorpion venom. Mass spectrometry-based proteomic techniques are commonly used in the study of scorpion venoms. The implementation of top-down and bottom-up approaches for proteomic analyses facilitates screening by allowing a global view of the structural aspects of such complex matrices. Here, we provide a partial overview of the venom of B. occitanus scorpion, in order to explore the diversity of its toxins and hereafter understand their effects. To this end, a combination of top-down and bottom-up approaches was applied using nano-high liquid chromatography coupled to nano-electrospray tandem mass spectrometry (nano-LC-ESI MS/MS). The LC-MS results showed that B. occitanus venom contains around 200 molecular masses ranging from 1868 to 16 720 Da, the most representative of which are those between 5000 and 8000 Da. Interestingly, combined top-down and bottom-up LC-MS/MS results allowed the identification of several toxins, which were mainly those acting on ion channels, including those targeting sodium (NaScTxs), potassium (KScTxs), chloride (ClScTxs), and calcium channels (CaScTx), as well as antimicrobial peptides (AMPs), amphipathic peptides, myotropic neuropeptides, and hypothetical secreted proteins. This study reveals the molecular diversity of B. occitanus scorpion venom and identifies components that may have useful pharmacological activities.

Optimization of a Top-Down Proteomics Platform for Closely Related Pathogenic Bacterial Discrimination.

The current technique used for microbial identification in hospitals is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, it suffers from important limitations, in particular, for closely related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a liquid chromatography (LC)-MS/MS top-down proteomics platform, which aims at discriminating closely related pathogenic bacteria through the identification of specific proteoforms. Using Escherichia coli as a model, all steps of the workflow were optimized: protein extraction, on-line LC separation, MS method, and data analysis. Using optimized parameters, about 220 proteins, corresponding to more than 500 proteoforms, could be identified in a single run. We then used this platform for the discrimination of enterobacterial pathogens undistinguishable by MALDI-TOF, although leading to very different clinical outcomes. For each pathogen, we identified specific proteoforms that could potentially be used as biomarkers. We also improved the characterization of poorly described bacterial strains. Our results highlight the advantage of addressing proteoforms rather than peptides for accurate bacterial characterization and qualify top-down proteomics as a promising tool in clinical microbiology. Data are available via ProteomeXchange with the identifier PXD019247.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Defective lytic transglycosylase disrupts cell morphogenesis by hindering cell wall de-O-acetylation in Neisseria meningitidis.

Lytic transglycosylases (LT) are enzymes involved in peptidoglycan (PG) remodeling. However, their contribution to cell-wall-modifying complexes and their potential as antimicrobial drug targets remains unclear. Here, we determined a high-resolution structure of the LT, an outer membrane lipoprotein from Neisseria species with a disordered active site helix (alpha helix 30). We show that deletion of the conserved alpha-helix 30 interferes with the integrity of the cell wall, disrupts cell division, cell separation, and impairs the fitness of the human pathogen Neisseria meningitidis during infection. Additionally, deletion of alpha-helix 30 results in hyperacetylated PG, suggesting this LtgA variant affects the function of the PG de-O-acetylase (Ape 1). Our study revealed that Ape 1 requires LtgA for optimal function, demonstrating that LTs can modulate the activity of their protein-binding partner. We show that targeting specific domains in LTs can be lethal, which opens the possibility that LTs are useful drug-targets.

Bacteria are surrounded by a tough yet flexible wall that protects the cell and serves as an anchor for several of the cell's structures. This cell wall contains a large mesh-like molecule called peptidoglycan made of many repeated building blocks. When a bacterial cell divides in two, it needs to make more of this material. Making peptidoglycan involves two different sets of enzymes working together: "polymerases" are the enzymes that link the individual building blocks to peptidoglycan, one after the other; while "lytic transglycosylases" are enzymes that modify the peptidoglycan to create space for the addition of new building blocks and for assemblies of proteins that must span the cell wall. Lytic transglycosylases are known to assemble with other proteins and enzymes to form the cell's peptidoglycan-modifying machinery, but it was not clear exactly what purpose they serve within these "enzyme complexes". It was also unclear whether these enzymes would be good targets for new antibiotics. To help answer these questions, Williams et al. looked at a lytic transglycoslyase called LtgA. This enzyme is originally from Neisseria meningitidis, a bacterium that can cause meningitis and life-threatening sepsis in humans. Williams et al. discovered that part of the enzyme's active site ­ the region of an enzyme where the chemical reaction takes ­ can switch from an ordered helix to a disordered, flexible loop. Bacteria were then genetically engineered to make a version of the enzyme that lacked this helix. These bacteria had weaker cell walls and were deformed; they were also less able to grow and divide, both in the laboratory and in a mouse model of infection. Further analysis showed that the deletion of the helix from the enzyme resulted in the peptidoglycan being modified much more than normal, which could likely explain their reduced virulence. Williams et al. also found that deleting the helix from LtgA interfered with the activity of a protein that interacts with this enzyme, called Ape1, which also contributed to the fragility of the cell wall. This shows that lytic transglycosylases assembled into enzyme complexes can alter the activities of other proteins in the complex. Together these findings show that researchers could target one enzyme in a complex in bacteria, and disrupt the activity of other proteins in that complex. This highlights the possibility of considering enzyme complexes as useful targets for new drugs, which is important considering the current problem of antibiotic resistance.

Biogenesis and structure of a type VI secretion baseplate.

To support their growth in a competitive environment and cause pathogenesis, bacteria have evolved a broad repertoire of macromolecular machineries to deliver specific effectors and toxins. Among these multiprotein complexes, the type VI secretion system (T6SS) is a contractile nanomachine that targets both prokaryotic and eukaryotic cells. The T6SS comprises two functional subcomplexes: a bacteriophage-related tail structure anchored to the cell envelope by a membrane complex. As in other contractile injection systems, the tail is composed of an inner tube wrapped by a sheath and built on the baseplate. In the T6SS, the baseplate is not only the tail assembly platform, but also docks the tail to the membrane complex and hence serves as an evolutionary adaptor. Here we define the biogenesis pathway and report the cryo-electron microscopy (cryo-EM) structure of the wedge protein complex of the T6SS from enteroaggregative Escherichia coli (EAEC). Using an integrative approach, we unveil the molecular architecture of the whole T6SS baseplate and its interaction with the tail sheath, offering detailed insights into its biogenesis and function. We discuss architectural and mechanistic similarities but also reveal key differences with the T4 phage and Mu phage baseplates.

Characterization of LE3 and LE4, the only lytic phages known to infect the spirochete Leptospira.

Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira.

Glycosylphosphatidylinositol Anchors from Galactomannan and GPI-Anchored Protein Are Synthesized by Distinct Pathways in Aspergillus fumigatus.

Glycosylphosphatidylinositols (GPIs) are lipid anchors allowing the exposure of proteins at the outer layer of the plasma membrane. In fungi, a number of GPI-anchored proteins (GPI-APs) are involved in the remodeling of the cell wall polymers. GPIs follow a specific biosynthetic pathway in the endoplasmic reticulum. After the transfer of the protein onto the GPI-anchor, a lipid remodeling occurs to substitute the diacylglycerol moiety by a ceramide. In addition to GPI-APs, A. fumigatus produces a GPI-anchored polysaccharide, the galactomannan (GM), that remains unique in the fungal kingdom. To investigate the role of the GPI pathway in the biosynthesis of the GM and cell wall organization, the deletion of PER1-coding for a phospholipase required for the first step of the GPI lipid remodeling-was undertaken. Biochemical characterization of the GPI-anchor isolated from GPI-APs showed that the PER1 deficient mutant produced a lipid anchor with a diacylglycerol. The absence of a ceramide on GPI-anchors in the Δper1 mutant led to a mislocation of GPI-APs and to an alteration of the composition of the cell wall alkali-insoluble fraction. On the other hand, the GM isolated from the Δper1 mutant membranes possesses a ceramide moiety as the parental strain, showing that GPI anchor of the GM follow a distinct unknown biosynthetic pathway.

A step-by-step in crystallo guide to bond cleavage and 1,6-anhydro-sugar product synthesis by a peptidoglycan-degrading lytic transglycosylase.

Lytic transglycosylases (LTs) are a class of enzymes important for the recycling and metabolism of peptidoglycan (PG). LTs cleave the ß-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc in the PG glycan strand, resulting in the concomitant formation of 1,6-anhydro-N-acetylmuramic acid and GlcNAc. No LTs reported to date have utilized chitins as substrates, despite the fact that chitins are GlcNAc polymers linked via ß-1,4-glycosidic bonds, which are the known site of chemical activity for LTs. Here, we demonstrate enzymatically that LtgA, a non-canonical, substrate-permissive LT from Neisseria meningitidis utilizes chitopentaose ((GlcNAc)5) as a substrate to produce three newly identified sugars: 1,6-anhydro-chitobiose, 1,6-anhydro-chitotriose, and 1,6-anhydro-chitotetraose. Although LTs have been widely studied, their complex reactions have not previously been visualized in the crystalline state because macromolecular PG is insoluble. Here, we visualized the cleavage of the glycosidic bond and the liberation of GlcNAc-derived residues by LtgA, followed by the synthesis of atypical 1,6-anhydro-GlcNAc derivatives. In addition to the newly identified anhydro-chitin products, we identified trapped intermediates, unpredicted substrate rearrangements, sugar distortions, and a conserved crystallographic water molecule bound to the catalytic glutamate of a high-resolution native LT. This study enabled us to propose a revised alternative mechanism for LtgA that could also be applicable to other LTs. Our work contributes to the understanding of the mechanisms of LTs in bacterial cell wall biology.

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough.

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.

Stability, structural and functional properties of a monomeric, calcium-loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins.

Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform.

Type II secretion systems (T2SSs) promote secretion of folded proteins playing important roles in nutrient acquisition, adaptation and virulence of Gram-negative bacteria. Protein secretion is associated with the assembly of type 4 pilus (T4P)-like fibres called pseudopili. Initially membrane embedded, pseudopilin and T4 pilin subunits share conserved transmembrane segments containing an invariant Glu residue at the fifth position, E5. Mutations of E5 in major T4 pilins and in PulG, the major pseudopilin of the Klebsiella T2SS abolish fibre assembly and function. Among the four minor pseudopilins, only PulH required E5 for secretion of pullulanase, the substrate of the Pul T2SS. Mass-spectrometry analysis of pili resulting from the co-assembly of PulG(E5A) variant and PulG(WT) ruled out an E5 role in pilin processing and N-methylation. A bacterial two-hybrid analysis revealed interactions of the full-length pseudopilins PulG and PulH with the PulJ-PulI-PulK priming complex and with the assembly factors PulM and PulF. Remarkably, PulG(E5A) and PulH(E5A) variants were defective in interaction with PulM but not with PulF, and co-purification experiments confirmed the E5-dependent interaction between native PulM and PulG. These results reveal the role of E5 in a recruitment step critical for assembly of the functional T2SS, likely relevant to T4P assembly systems.