Candidalysin is a fungal peptide toxin critical for mucosal infection.

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA.

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.

Mode of Action of the Catalytic Site in the N-Terminal Ribosome-Inactivating Domain of JIP60.

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.

Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein.

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2.

The T7 phage-encoded small protein Gp2 is a non-DNA-binding transcription factor that interacts with the jaw domain of the Escherichia coli (Ec) RNA polymerase (RNAp) ß' subunit and inhibits transcriptionally proficient promoter-complex (RPo) formation. Here, we describe the high-resolution solution structure of the Gp2-Ec ß' jaw domain complex and show that Gp2 and DNA compete for binding to the ß' jaw domain. We reveal that efficient inhibition of RPo formation by Gp2 requires the amino-terminal σ(70) domain region 1.1 (R1.1), and that Gp2 antagonizes the obligatory movement of R1.1 during RPo formation. We demonstrate that Gp2 inhibits RPo formation not just by steric occlusion of the RNAp-DNA interaction but also through long-range antagonistic effects on RNAp-promoter interactions around the RNAp active center that likely occur due to repositioning of R1.1 by Gp2. The inhibition of Ec RNAp by Gp2 thus defines a previously uncharacterized mechanism by which bacterial transcription is regulated by a viral factor.

The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain.

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

Structural insight into host recognition by aggregative adherence fimbriae of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.

The peptide-binding cavity is essential for Als3-mediated adhesion of Candida albicans to human cells.

The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion.

Structural insights into the biogenesis and biofilm formation by the Escherichia coli common pilus.

Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.

Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.

Candida albicans is the most prevalent fungal pathogen in humans and a major source of life-threatening nosocomial infections. The Als (agglutinin-like sequence) glycoproteins are an important virulence factor for this fungus and have been associated with binding of host-cell surface proteins and small peptides of random sequence, the formation of biofilms and amyloid fibers. High-resolution structures of N-terminal Als adhesins (NT-Als; up to 314 amino acids) show that ligand recognition relies on a motif capable of binding flexible C termini of peptides in extended conformation. Central to this mechanism is an invariant lysine that recognizes the C-terminal carboxylate of ligands at the end of a deep-binding cavity. In addition to several protein-peptide interactions, a network of water molecules runs parallel to one side of the ligand and contributes to the recognition of diverse peptide sequences. These data establish NT-Als adhesins as a separate family of peptide-binding proteins and an unexpected adhesion system for primary, widespread protein-protein interactions at the Candida/host-cell interface.

A RabGAP negatively regulates plant autophagy and immune trafficking.

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as a key membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL reduces autophagic flux in three model plant species, suggesting that its negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

Regulation of the Plasmodium motor complex: phosphorylation of myosin A tail-interacting protein (MTIP) loosens its grip on MyoA.

The interaction between the C-terminal tail of myosin A (MyoA) and its light chain, myosin A tail domain interacting protein (MTIP), is an essential feature of the conserved molecular machinery required for gliding motility and cell invasion by apicomplexan parasites. Recent data indicate that MTIP Ser-107 and/or Ser-108 are targeted for intracellular phosphorylation. Using an optimized MyoA tail peptide to reconstitute the complex, we show that this region of MTIP is an interaction hotspot using x-ray crystallography and NMR, and S107E and S108E mutants were generated to mimic the effect of phosphorylation. NMR relaxation experiments and other biophysical measurements indicate that the S108E mutation serves to break the tight clamp around the MyoA tail, whereas S107E has a smaller but measurable impact. These data are consistent with physical interactions observed between recombinant MTIP and native MyoA from Plasmodium falciparum lysates. Taken together these data support the notion that the conserved interactions between MTIP and MyoA may be specifically modulated by this post-translational modification.

T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the host RNA polymerase (RNAP)--a multi-subunit enzyme responsible for gene transcription--by a small ( approximately 7 kDa) phage-encoded protein called Gp2. Gp2 is also a potent inhibitor of E. coli RNAP in vitro. Here we describe the first atomic resolution structure of Gp2, which reveals a distinct run of surface-exposed negatively charged amino acid residues on one side of the molecule. Our comprehensive mutagenesis data reveal that two conserved arginine residues located on the opposite side of Gp2 are important for binding to and inhibition of RNAP. Based on a structural model of the Gp2-RNAP complex, we propose that inhibition of transcription by Gp2 involves prevention of RNAP-promoter DNA interactions required for stable DNA strand separation and maintenance of the "transcription bubble" near the transcription start site, an obligatory step in the formation of a transcriptionally competent promoter complex.

Structural insight into the role of Streptococcus parasanguinis Fap1 within oral biofilm formation.

The fimbriae-associated protein 1 (Fap1) is a major adhesin of Streptococcus parasanguinis, a primary colonizer of the oral cavity that plays an important role in the formation of dental plaque. Fap1 is an extracellular adhesive surface fibre belonging to the serine-rich repeat protein (SRRP) family, which plays a central role in the pathogenesis of streptococci and staphylococci. The N-terminal adhesive region of Fap1 (Fap1-NR) is composed of two domains (Fap1-NR(α) and Fap1-NR(ß)) and is projected away from the bacterial surface via the extensive serine-rich repeat region, for adhesion to the salivary pellicle. The adhesive properties of Fap1 are modulated through a pH switch in which a reduction in pH results in a rearrangement between the Fap1-NR(α) and Fap1-NR(ß) domains, which assists in the survival of S. parasanguinis in acidic environments. We have solved the structure of Fap1-NR(α) at pH 5.0 at 3.0Ǻ resolution and reveal how subtle rearrangements of the 3-helix bundle combined with a change in electrostatic potential mediates 'opening' and activation of the adhesive region. Further, we show that pH-dependent changes are critical for biofilm formation and present an atomic model for the inter-Fap1-NR interactions which have been assigned an important role in the biofilm formation.

Promoting crystallisation of the Salmonella enteritidis fimbriae 14 pilin SefD using deuterium oxide.

The use of heavy water (D(2)O) as a solvent is commonplace in many spectroscopic techniques for the study of biological macromolecules. A significant deuterium isotope effect exists where hydrogen-bonding is important, such as in protein stability, dynamics and assembly. Here we illustrate the use of D(2)O in additive screening for the production of reproducible diffraction-quality crystals for the Salmonella enteritidis fimbriae 14 (SEF14) putative tip adhesin, SefD.

Identification of the first structurally validated covalent ligands of the small GTPase RAB27A.

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein-protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket ('WF-binding pocket') via a conserved tryptophan-phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

Structural insights into serine-rich fimbriae from Gram-positive bacteria.

The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.

Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain.

The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatized crystals suitable for structural determination. Unlike in previous reports, a standard autotrophic expression strain was used and only single-wavelength anomalous dispersion (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. The native protein crystallized in two different crystal forms (form 1 belonging to space group C222(1) and form 2 belonging to space group C2), which diffracted to 2.4 and 2.0 Šresolution, respectively, whilst Se-derivatized protein crystallized in space group C2 and diffracted to 1.7 Šresolution. The Se-derivatized crystals are suitable for SAD structure determination using only the anomalous signal derived from the SeCys residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.

Expression, crystallization and preliminary X-ray data analysis of NT-Als9-2, a fungal adhesin from Candida albicans.

Candida albicans is a common human fungal commensal that can also cause a range of infections from skin/mucosal `thrush' to severe systemic candidiasis. Adherence to host cells is one of the key determinants of Candida pathogenesis. The Als family of surface proteins has been implicated in adhesion of C. albicans, yet limited information has been published on the structure and mechanism of these fungal adhesins. The N-terminal region of these proteins has been shown to possess adhesive properties, making it a possible target for new therapeutic strategies. Recombinant NT-Als9-2 from C. albicans (residues 18-329) was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.0 Šresolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.73, b = 68.71, c = 120.03 Å, α = ß = γ = 90° and one molecule in the asymmetric unit. Platinum-derivatized crystals belonged to the same space group, with similar unit-cell parameters, although they were not completely isomorphous.

Peptide Probes for Plasmodium falciparum MyoA Tail Interacting Protein (MTIP): Exploring the Druggability of the Malaria Parasite Motor Complex.

Malaria remains an endemic tropical disease, and the emergence of Plasmodium falciparum parasites resistant to current front-line medicines means that new therapeutic targets are required. The Plasmodium glideosome is a multiprotein complex thought to be essential for efficient host red blood cell invasion. At its core is a myosin motor, Myosin A (MyoA), which provides most of the force required for parasite invasion. Here, we report the design and development of improved peptide-based probes for the anchor point of MyoA, the P. falciparum MyoA tail interacting protein (PfMTIP). These probes combine low nanomolar binding affinity with significantly enhanced cell penetration and demonstrable competitive target engagement with native PfMTIP through a combination of Western blot and chemical proteomics. These results provide new insights into the potential druggability of the MTIP/MyoA interaction and a basis for the future design of inhibitors.