Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Reduced specificity and increased overgenerality of autobiographical memory persist as cognitive vulnerabilities in remitted major depression: A meta-analysis.

Difficulty in accessing specific memories, referred to as reduced memory specificity or overgeneral memory (OGM), has been established as a marker of clinical depression. However, it is not clear if this deficit persists following the remission of depressive episodes. The current study involved a systematic review and meta-analysis of empirical studies with the aim of establishing whether remitted depression was associated with retrieving fewer specific and more overgeneral autobiographical memories. Seventeen studies were identified as eligible. The results indicated that people with remitted depression recalled fewer specific memories (k = 15; g = -0.314, 95% CI [-0.543; -0.085], z = -2.69, p = .007) and more categoric memories (k = 9; g = 0.254, 95% CI [0.007; 0.501], z = 2.02, p = .043) compared to people who had never been depressed. Given these deficits have elsewhere been shown to be prognostic of future depressive symptoms, these findings suggest that reduced memory specificity/overgeneral memory persists following remission and may be a risk factor for future episodes of depression in those that are in remission. The findings are discussed in terms of how this knowledge might influence clinical understanding of relapse prevention and maintenance of remission in those with a history of depression.

Molecular structure of the intact bacterial flagellar basal body.

The bacterial flagellum is a macromolecular protein complex that enables motility in many species. Bacterial flagella self-assemble a strong, multicomponent drive shaft that couples rotation in the inner membrane to the micrometre-long flagellar filament that powers bacterial swimming in viscous fluids1-3. Here, we present structures of the intact Salmonella flagellar basal body4, encompassing the inner membrane rotor, drive shaft and outer-membrane bushing, solved using cryo-electron microscopy to resolutions of 2.2-3.7 Å. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the rotor component with both the export gate complex and the proximal rod forming interactions with the MS-ring. At the other end, the drive shaft distal rod passes through the LP-ring bushing complex, which functions as a molecular bearing anchored in the outer membrane through interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insights into the assembly process of this molecular machine.

The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion.

Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins (FliPQR in flagella, SctRST in virulence systems). A fourth essential T3SS protein (FlhB/SctU) functions to "switch" secretion substrate specificity once the growing hook/needle reach their determined length. Here, we present the cryo-electron microscopy structure of an export gate containing the switch protein from a Vibrio flagellar system at 3.2 Å resolution. The structure reveals that FlhB/SctU extends the helical export gate with its four predicted transmembrane helices wrapped around FliPQR/SctRST. The unusual topology of the FlhB/SctU helices creates a loop wrapped around the bottom of the closed export gate. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the T3SS trigger opening of the gate through interactions between FlhB/SctU and FliPQR/SctRST.

FHR-1 Binds to C-Reactive Protein and Enhances Rather than Inhibits Complement Activation.

Factor H-related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

Different modes of variation for each BG lineage suggest different functions.

Mammalian butyrophilins have various important functions, one for lipid binding but others as ligands for co-inhibition of αß T cells or for stimulation of γδ T cells in the immune system. The chicken BG homologues are dimers, with extracellular immunoglobulin variable (V) domains joined by cysteines in the loop equivalent to complementarity-determining region 1 (CDR1). BG genes are found in three genomic locations: BG0 on chromosome 2, BG1 in the classical MHC (the BF-BL region) and many BG genes in the BG region just outside the MHC. Here, we show that BG0 is virtually monomorphic, suggesting housekeeping function(s) consonant with the ubiquitous tissue distribution. BG1 has allelic polymorphism but minimal sequence diversity, with the few polymorphic residues at the interface of the two V domains, suggesting that BG1 is recognized by receptors in a conserved fashion. Any phenotypic variation should be due to the intracellular region, with differential exon usage between alleles. BG genes in the BG region can generate diversity by exchange of sequence cassettes located in loops equivalent to CDR1 and CDR2, consonant with recognition of many ligands or antigens for immune defence. Unlike the mammalian butyrophilins, there are at least three modes by which BG genes evolve.

Cytomegalovirus-associated thrombocytopenia treated with thrombopoietin receptor agonist.

Symptomatic cytomegalovirus (CMV) in immunocompetent individuals is an uncommon, yet recognised, phenomenon. This report discusses a case of CMV-associated thrombocytopenia refractory to immunomodulation and antivirals. Ultimately, a clinicopathological response was attained with eltrombopag. This is the first report of the efficacy of thrombopoietin receptor agonists in this context. This experience provides insight into possible pathogenic mechanisms of CMV-associated thrombocytopenia and presents a therapeutic option.

Resolution of paraneoplastic immune thrombocytopenia following everolimus treatment for metastatic renal cell carcinoma.

Autoimmune thrombocytopenia is an uncommon but reported paraneoplastic manifestation of renal cell carcinoma (RCC). Treatment usually involves management of the underlying malignancy; however, steroids have shown a benefit in published case reports. Here, we describe a patient with profound thrombocytopenia secondary to metastatic RCC. It was refractory to steroid and intravenous immunoglobulin, but the platelet count improved markedly following initiation of everolimus. The possible explanation includes immunomodulation, tumour lysis or a combination of both effects. This is the first reported case of everolimus used in paraneoplastic thrombocytopenia from RCC. More studies are needed for further investigation of its potential use in secondary immune thrombocytopenia from RCC and perhaps other malignancies.

Factor H-related protein 5 interacts with pentraxin 3 and the extracellular matrix and modulates complement activation.

The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.

Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility.

Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease.

Infusion of foam sclerosants results in a distance-dependent procoagulant activity, haemoconcentration and elevation of D-dimer levels.

OBJECTIVE: To investigate the biological effects of foam sclerotherapy in vivo. MATERIALS AND METHODS: Ultrasound-guided sclerotherapy was performed using a 3% sodium tetradecyl sulphate or polidocanol. A total of 15 mL of foam was injected. Samples were collected from antecubital veins, target saphenous veins and the adjoining deep veins before, immediately after and 1 hour after the procedure. Saphenous vein samples were also taken sequentially at set 15 cm intervals. Clotting times, D-dimer, cell counts and biochemical parameters were measured. D-dimer levels were repeated one week later. RESULTS: Forty procedures were performed. Systemic clotting times were not affected by the procedure. Injection of 0.5 mL of foam 5 cm away from the relevant junctions resulted in procoagulant activity in the adjoining deep veins (sodium tetradecyl sulphate) and the target saphenous veins (sodium tetradecyl sulphate and polidocanol). The procoagulant effect in the target veins reached a peak at 15 cm but normalised at 45 cm. D-dimer levels were significantly increased 1 hour after treatment with either agent and remained elevated one week later. Sodium tetradecyl sulphate and to a lesser degree polidocanol induced biochemical changes consistent with haemoconcentration. CONCLUSION: Infusion of foam sclerosants results in a distance-dependent procoagulant activity in the exposed vessels. Foam sclerotherapy results in haemoconcentration and elevation of D-dimer.

Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

ErpC, a member of the complement regulator-acquiring family of surface proteins from Borrelia burgdorferi, possesses an architecture previously unseen in this protein family.

Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts.

Further structural insights into the binding of complement factor H by complement regulator-acquiring surface protein 1 (CspA) of Borrelia burgdorferi.

Borrelia burgdorferi has evolved many mechanisms of evading the different immune systems across its range of reservoir hosts, including the capture and presentation of host complement regulators factor H and factor H-like protein-1 (FHL-1). Acquisition is mediated by a family of complement regulator-acquiring surface proteins (CRASPs), of which the atomic structure of CspA (BbCRASP-1) is known and shows the formation of a homodimeric species which is required for binding. Mutagenesis studies have mapped a putative factor H binding site to a cleft between the two subunits. Presented here is a new atomic structure of CspA which shows a degree of flexibility between the subunits which may be critical for factor H scavenging by increasing access to the binding interface and allows the possibility that the assembly can clamp around the bound complement regulators.

Bifunctional lipocalin ameliorates murine immune complex-induced acute lung injury.

Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics. OmCI is a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB4) within an internal binding pocket. Here, we examined the effect of LTB4 binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB4 in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB4 (C20). We show that the C5 and LTB4 binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB4 binding for full activity. The data highlight the importance of LTB4 in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB4 may be useful for treatment of human immune complex-dependent diseases.

Dimerization of complement factor H-related proteins modulates complement activation in vivo.

The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Investigating the structure of the factor B vWF-A domain/CD55 protein-protein complex using DEER spectroscopy: successes and pitfalls.

The electron paramagnetic resonance technique of double electron-electron resonance (DEER) was used to measure nanometre-scale distances between nitroxide spin labels attached to the complement regulatory protein CD55 (also known as decay accelerating factor) and the von Willebrand factor A (vWF-A) domain of factor B. Following a thorough assessment of the quality of the data, distances obtained from good-quality measurements are compared to predicted distances from a previously hypothesised model for the complex and are found to be incompatible. The success of using these distances as restraints in multi-body docking routines is presented critically.

Structures of the rat complement regulator CrrY.

Complement receptor 1-related protein Y (CrrY) is an important cell-surface regulator of complement that is unique to rodent species. The structure of rat CrrY domains 1-4 has been determined in two distinct crystal forms and reveals a 70° bend between domains 3 and 4. Comparisons of this structure with those of other complement regulators suggests that rearrangement of this interface may occur on forming the regulatory complex with C3b.

Structural basis for complement factor I control and its disease-associated sequence polymorphisms.

The complement system is a key component of innate and adaptive immune responses. Complement regulation is critical for prevention and control of disease. We have determined the crystal structure of the complement regulatory enzyme human factor I (fI). FI is in a proteolytically inactive form, demonstrating that it circulates in a zymogen-like state despite being fully processed to the mature sequence. Mapping of functional data from mutants of fI onto the structure suggests that this inactive form is maintained by the noncatalytic heavy-chain allosterically modulating activity of the light chain. Once the ternary complex of fI, a cofactor and a substrate is formed, the allosteric inhibition is released, and fI is oriented for cleavage. In addition to explaining how circulating fI is limited to cleaving only C3b/C4b, our model explains the molecular basis of disease-associated polymorphisms in fI and its cofactors.

In vitro effects of detergent sclerosants on clot formation and fibrinolysis.

OBJECTIVE: To investigate the in vitro effects of detergent sclerosants sodium tetradecyl sulphate (STS) and polidocanol (POL) on clot formation and lysis. MATERIALS AND METHODS: clot kinetics were assessed in whole blood by thromboelastography (TEG®) and rotational thromboelastometry (ROTEM®). Fibrinogen was measured by the Clauss method in plasma and factor XIII (FXIII) by enzyme-linked immunosorbent assay (ELISA). Turbidity measurements were used to assess clot lysis in plasma, and fibrinolysis in non-cross-linked and cross-linked fibrin. D-dimer was measured by VIDAS®, STA®Liatest® and AxSYM® assays. RESULTS: Strong clots were formed at low sclerosant concentrations (0.075-0.1%). At midrange concentrations (0.15% STS, 0.15-0.3% POL), both agents inhibited the contribution of platelets to clot firmness and formed weak clots prone to lysis. At higher concentrations (STS ≥ 0.3% and POL ≥ 0.6%), clot formation was inhibited. STS destroyed FXIII at ≥ 0.15% and fibrinogen at ≥ 0.6%. Neither sclerosant had a significant effect on cross-linked fibrin, but STS had a lytic effect on non-cross-linked fibrin. STS caused an artefactual elevation of D-dimer in the VIDAS® assay when fibrinogen was present. CONCLUSION: Detergent sclerosants demonstrated a trimodal effect on clot formation, initiating strong clots at low concentrations, weak clots at midrange concentrations and preventing clot formation at higher concentrations. Neither agent had fibrinolytic activity.