Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.

Background: Of the eight cell surface glycoproteins in the C. albicans Als family, Als3 makes the largest contribution to adhesion to human cells.Results: Mutation of the Als3 peptide-binding cavity (PBC) results in loss of Als3 adhesive function.Conclusion: The PBC is required for Als3 adhesive function.Significance: Interfering with PBC function is a viable strategy for inhibiting C. albicans adhesion.

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.

New direct acting antivirals (DAAs) such as daclatasvir (DCV; BMS-790052), which target NS5A function with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however, major resistance mutations appear in the N-terminal region of NS5A that include the amphipathic helix and domain 1. The dimeric symmetry of these compounds suggests that they act on a dimer of NS5A, which is also consistent with the presence of dimers in crystals of NS5A domain 1 from genotype 1b. Genotype 1a HCV is less potently affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A homologue and intriguingly, our X-ray crystal structure reveals two new dimeric forms of this domain. Furthermore, the high solvent content (75%) makes it ideal for ligand-soaking. Daclatasvir (DCV) shows twofold symmetry suggesting NS5A dimers may be of physiological importance and serve as potential binding sites for DCV. These dimers also allow for new conformations of a NS5A expansive network which could explain its operation on the membranous web. Additionally, sulfates bound in the crystal structure may provide evidence for the previously proposed RNA binding groove, or explain regulation of NS5A domain 2 and 3 function and phosphorylation, by domain 1.

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.

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.chaperone-usher | donor-strand exchange | X-ray crystallography T he long coexistence between bacteria and their eukaryote hosts has fine-tuned their relationships. These microorganisms have developed remarkable molecular systems for the trafficking of molecules across the cell envelope and exchanging signals with their environment. Historically, bacteria have been studied during their planktonic life cycle, although a matrix-enclosed, organized-community lifestyle is a frequent mode of growth for countless species that exist in diverse environments. Many bacteria produce surface fibers allowing them to adhere to each other and to biotic and abiotic surfaces (1, 2). Furthermore, many of these fibers are able to recognize specific host cell receptors during the first steps of host colonization and subsequent biofilm formation (3). These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease allowing reservoirs of bacteria to persist in a host or the environment. Biofilms also contribute to increased resistance to antibiotics, the immune system, and host clearance mechanisms (4).Escherichia coli are primarily commensal colonizers of the human and other animal bowels, contributing to a healthy host immune system. Conversely, there are a number of virulent strains that are important causative agents of many diarrheal diseases such as hemorrhagic colitis (4). Upon entering extraintestinal sites, pathogenic strains can also lead to neonatal meningitis, urinary tract infections, sepsis, and pneumonia (5). The effects of such gastrointestinal and urinary tract diseases in the developing world are considerable (6), and they are estimated to kill around 2.5 million people every year, mostly young children (7).The E. coli common pilus (ECP), encoded by the ecpRABCDE operon, is an extracellular adhesive fiber first documented in association with E. coli strains causing newborn meningitis and septicaemia (NMEC), where it was originally named the Mat (meningitis-associated and temperature-regulated) fimbriae (8). Furthe...

Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce "the trigger mechanism of cell entry." Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.Colonization, multiplication, and dissemination are the key steps of an infectious cycle. To persist within the hostile in vivo environments, bacterial pathogens utilize sophisticated virulence strategies to subvert and hijack cellular and systemic functions. A common infection strategy used by Gram-negative pathogens involves injection of virulence factors, known as effectors, by the type III secretion system (T3SS) from the bacterial cell directly into the eukaryotic cell (reviewed in reference 26). The injected effectors target different cellular compartments and subvert numerous signaling pathways for the benefit of the invaded or attached bacteria. Due to their essential role in the regulation of key cellular functions, Rho family small G proteins are common targets of T3SS bacterial effectors (reviewed in reference 23).To date, 22 members of the Rho GTPase family, belonging to the small GTPase protein superfamily, have been identified (54). Cdc42, Rac1, and RhoA, which trigger formation of filopodia, lamellipodia/ruffles, and stress fibers, respectively, are the best characterized (29). The Rho GTPases share a conserved structure, consisting of two flexible domains called switch I and switch II and a phosphate binding loop (P-loop), which together form a Mg 2ϩ -and nucleotide-binding pocket (reviewed in reference 22). The small GTPases are modified posttranscriptionally by the addition of a lipid moiety to the C terminus (farnesyl, geranyl, palmitoyl, or methyl), signaled by the carboxy-terminal CAAX motif (55), which targets them to different membranous compartments.The function of the small GTPases is strictly regulated. By binding the two switch domains and the lipid moiety, the guanine nucleotide dissociation inhibitors (GDIs) prevent membrane localizatio...

The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1–SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops.