Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

Membrane proteins of Pasteurella multocida : roles in immunity & disease

The cell surface protects Gram-negative bacteria against a range of harsh environments and is critical for interaction of the bacterium the host cells and tissues. The outer membrane (OM) functions as a selective barrier that prevents the entry of many toxic molecules into the cell, a property that is crucial for bacterial survival in many environments. At the same time, the embedded proteins in the outer membrane fulfil a number of tasks that are crucial to the bacterial cell, such as nutrient uptake, transport of various molecules in and out of the cell, and interaction with host tissues. Pathogens have evolved different types of transport systems that contribute to their survival and dissemination within the host. The pumps that effect efflux, which include members of the TolC family, typically export several unrelated substances, including antibiotics, organic solvents, and bile produced by the host. While much data exist vis-a-vis the underlying mechanisms of multidrug efflux pumps in bacteria, in Pasteurella multocida this remains unknown. In this study, we characterised two outer membrane proteins, encoded by the genes pm0527 and pm1980, predicted to be TolC homologues in P. multocida. Compared to the wild type, the pm0527 mutant showed up to a 512-fold increase in susceptibility to a range of antibiotics and other chemical agents. Complementation of the mutant with an intact pm0527 gene restored the resistance to gentamicin, novobiocin and numerous other compounds. The pm1980 showed a 64-fold increase in susceptibility to rifampin. Together, these findings provide strong evidence that PM0527 and PM1980 function as components of multidrug efflux pumps that contribute to the intrinsic resistance of P. multocida to a broad range of structurally unrelated antibacterial agents. Methionine is an essential amino acid for all living organisms. However, in bacteria no prior evidence has been reported to substantiate this claim in vivo. Here, we characterised a predicted methionine-binding lipoprotein, PlpB, and investigated its role in the pathogenesis of P. multocida. Inactivation of the plpB gene in P. multocida resulted in full attenuation in chickens, as assessed by both direct challenge and competitive in vivo growth assays. Virulence was restored by complementation with an intact plpB gene. In vitro biochemical analyses using radiolabelled amino acids demonstrated that soluble recombinant PlpB can bind both L- and D-methionine. Inhibition studies showed that non-labelled L-and D-methionine could both compete for binding with their labelled counterparts. These results suggest that PlpB is a novel, virulence-associated factor that is involved in the survival of P. multocida in chickens. Bioinformatics and structural studies of PlpB in comparison to the crystal structure of a related protein, Tp32, show that 8 of the 10 active site residues that interact with the methionine residue are identical, one is conserved, and the other one is a non-conserved substitution. In this work we report the vaccination results of five recombinant OM proteins of P. multocida. Out of the five proteins tested, the urea-solubilised recombinant PlpE showed protection in mice and chickens. Additional experiments in chickens and mice using insertionally inactivated mutants of all six genes showed that none of the encoded proteins played a significant role in the pathogenesis of P. multocida. Characterisation of PlpE by immunofluorescence microscopy showed it to be surface localised. This is the first report where a denatured recombinant protein, PlpE, has been shown to elicit protection against fowl cholera

Membrane proteins of Pasteurella multocida : roles in immunity & disease

The cell surface protects Gram-negative bacteria against a range of harsh environments and is critical for interaction of the bacterium the host cells and tissues. The outer membrane (OM) functions as a selective barrier that prevents the entry of many toxic molecules into the cell, a property that is crucial for bacterial survival in many environments. At the same time, the embedded proteins in the outer membrane fulfil a number of tasks that are crucial to the bacterial cell, such as nutrient uptake, transport of various molecules in and out of the cell, and interaction with host tissues. Pathogens have evolved different types of transport systems that contribute to their survival and dissemination within the host. The pumps that effect efflux, which include members of the TolC family, typically export several unrelated substances, including antibiotics, organic solvents, and bile produced by the host. While much data exist vis-a-vis the underlying mechanisms of multidrug efflux pumps in bacteria, in Pasteurella multocida this remains unknown. In this study, we characterised two outer membrane proteins, encoded by the genes pm0527 and pm1980, predicted to be TolC homologues in P. multocida. Compared to the wild type, the pm0527 mutant showed up to a 512-fold increase in susceptibility to a range of antibiotics and other chemical agents. Complementation of the mutant with an intact pm0527 gene restored the resistance to gentamicin, novobiocin and numerous other compounds. The pm1980 showed a 64-fold increase in susceptibility to rifampin. Together, these findings provide strong evidence that PM0527 and PM1980 function as components of multidrug efflux pumps that contribute to the intrinsic resistance of P. multocida to a broad range of structurally unrelated antibacterial agents. Methionine is an essential amino acid for all living organisms. However, in bacteria no prior evidence has been reported to substantiate this claim in vivo. Here, we characterised a predicted methionine-binding lipoprotein, PlpB, and investigated its role in the pathogenesis of P. multocida. Inactivation of the plpB gene in P. multocida resulted in full attenuation in chickens, as assessed by both direct challenge and competitive in vivo growth assays. Virulence was restored by complementation with an intact plpB gene. In vitro biochemical analyses using radiolabelled amino acids demonstrated that soluble recombinant PlpB can bind both L- and D-methionine. Inhibition studies showed that non-labelled L-and D-methionine could both compete for binding with their labelled counterparts. These results suggest that PlpB is a novel, virulence-associated factor that is involved in the survival of P. multocida in chickens. Bioinformatics and structural studies of PlpB in comparison to the crystal structure of a related protein, Tp32, show that 8 of the 10 active site residues that interact with the methionine residue are identical, one is conserved, and the other one is a non-conserved substitution. In this work we report the vaccination results of five recombinant OM proteins of P. multocida. Out of the five proteins tested, the urea-solubilised recombinant PlpE showed protection in mice and chickens. Additional experiments in chickens and mice using insertionally inactivated mutants of all six genes showed that none of the encoded proteins played a significant role in the pathogenesis of P. multocida. Characterisation of PlpE by immunofluorescence microscopy showed it to be surface localised. This is the first report where a denatured recombinant protein, PlpE, has been shown to elicit protection against fowl cholera

The regulative effect of Urtica dioica on sex hormones imbalance: elevated follicle-stimulating hormone/luteinizing hormone ratio ≥4.5 is associated with low performance in aged breeder quails

The age-related reproductive disorders are the main concerns in old birds. It was suggested that a drop in egg production and reproductive performance, towards the end of their laying period was caused partly by a decrease in the baseline concentration of plasma LH. Urtica dioica (nettle) is a plant with natural aromatase inhibitors. Steroid hormone levels are regulated by inhibition of the aromatase enzyme. Few studies have examined the effect of nettle on the egg production in adult hens. The aim of this study was to investigate the effects of diet supplemented with nettle powder (NP) in aged quails. One hundred and forty-52-week-old Japanese quails were randomly assigned to four treatments consisting of seven replicates (n = 5; four females and one male) and fed with diets containing NP at 0% (control group), 0.5, 1.0, and 1.5% (treatment groups). At 62 week of age, our results indicated the NP improved egg production, feed conversion ratio, eggshell thickness and Haugh unit (p < .05). Notably, fertility and hatchability of fertile eggs were significantly increased, while total embryonic mortality decreased significantly by supplementing diet with nettle powder (p < .05). Higher luteinizing hormone, lower oestrogen, malondialdehyde and total cholesterol and triglyceride concentrations were associated with percent of the nettle powder in diet (p ≤ .05). Elevated follicle-stimulating hormone/luteinizing hormone ratio ≥4 is associated with low egg production in control group and nettle supplementation can balance FSH/LH ratio to ≤2.7. It is concluded that nettle powder could be used as a worthwhile feed additive at the late laying period of aged quails.HIGHLIGHTS Nettle powder can be used as a food additive with aged quails at late laying period. Addition of 1 and 1.5% nettle powder improves egg production, FCR, egg shell thickness. Nettle enhances reproductive performances, such as fertility, hatchability, weight of ovary, and weight of follicles by balancing reproductive hormones at late laying period

Characterization of TolC Efflux Pump Proteins from Pasteurella multocida▿ †

Two TolC homologs, PM0527 and PM1980, were identified for Pasteurella multocida. A pm0527 mutant displayed increased susceptibility to a range of chemicals, including rifampin (512-fold) and acridine orange (128-fold). A pm1980 mutant showed increased susceptibility to rifampin, ceftazidime, and vancomycin

Comparative Surfaceome Analysis of Clonal <i>Histomonas meleagridis</i> Strains with Different Pathogenicity Reveals Strain-Dependent Profiles

Histomonas meleagridis, a poultry-specific intestinal protozoan parasite, is histomonosis’s etiological agent. Since treatment or prophylaxis options are no longer available in various countries, histomonosis can lead to significant production losses in chickens and mortality in turkeys. The surfaceome of microbial pathogens is a crucial component of host–pathogen interactions. Recent proteome and exoproteome studies on H. meleagridis produced molecular data associated with virulence and in vitro attenuation, yet the information on proteins exposed on the cell surface is currently unknown. Thus, in the present study, we identified 1485 proteins and quantified 22 and 45 upregulated proteins in the virulent and attenuated strains, respectively, by applying cell surface biotinylation in association with high-throughput proteomic analysis. The virulent strain displayed upregulated proteins that could be linked to putative virulence factors involved in the colonization and establishment of infection, with the upregulation of two candidates being confirmed by expression analysis. In the attenuated strain, structural, transport and energy production proteins were upregulated, supporting the protozoan’s adaptation to the in vitro environment. These results provide a better understanding of the surface molecules involved in the pathogenesis of histomonosis, while highlighting the pathogen’s in vitro adaptation processes

Screening of 71 P. multocida proteins for protective efficacy in a fowl cholera infection model and characterization of the protective antigen PlpE

Background: There is a strong need for a recombinant subunit vaccine against fowl cholera. We used a reverse vaccinology approach to identify putative secreted or cell surface associated P. multocida proteins that may represent potential vaccine candidate antigens. Principal Findings: A high-throughput cloning and expression protocol was used to express and purify 71 recombinant proteins for vaccine trials. Of the 71 proteins tested, only one, PlpE in denatured insoluble form, protected chickens against fowl cholera challenge. PlpE also elicited comparable levels of protection in mice. PlpE was localized by immunofluorescence to the bacterial cell surface, consistent with its ability to elicit a protective immune response. To explore the role of PlpE during infection and immunity, a plpE mutant was generated. The plpE mutant strain retained full virulence for mice. Conclusion: These studies show that PlpE is a surface exposed protein and was the only protein of 71 tested that was able to elicit a protective immune response. However, PlpE is not an essential virulence factor. This is the first report of a denatured recombinant protein stimulating protection against fowl cholera

The regulative effect of Urtica dioica on sex hormones imbalance: elevated follicle-stimulating hormone/luteinizing hormone ratio >= 4.5 is associated with low performance in aged breeder quails

The age-related reproductive disorders are the main concerns in old birds. It was suggested that a drop in egg production and reproductive performance, towards the end of their laying period was caused partly by a decrease in the baseline concentration of plasma LH. Urtica dioica (nettle) is a plant with natural aromatase inhibitors. Steroid hormone levels are regulated by inhibition of the aromatase enzyme. Few studies have examined the effect of nettle on the egg production in adult hens. The aim of this study was to investigate the effects of diet supplemented with nettle powder (NP) in aged quails. One hundred and forty-52-week-old Japanese quails were randomly assigned to four treatments consisting of seven replicates (n = 5; four females and one male) and fed with diets containing NP at 0% (control group), 0.5, 1.0, and 1.5% (treatment groups). At 62 week of age, our results indicated the NP improved egg production, feed conversion ratio, eggshell thickness and Haugh unit (p = 4 is associated with low egg production in control group and nettle supplementation can balance FSH/LH ratio to <= 2.7. It is concluded that nettle powder could be used as a worthwhile feed additive at the late laying period of aged quails

Design, Synthesis, and Biological Activity of 1,2,3-Triazolobenzodiazepine BET Bromodomain Inhibitors

A number of diazepines are known to inhibit bromo- and extra-terminal domain (BET) proteins. Their BET inhibitory activity derives from the fusion of an acetyl-lysine mimetic heterocycle onto the diazepine framework. Herein we describe a straightforward, modular synthesis of novel 1,2,3-triazolobenzodiazepines and show that the 1,2,3-triazole acts as an effective acetyl-lysine mimetic heterocycle. Structure-based optimization of this series of compounds led to the development of potent BET bromodomain inhibitors with excellent activity against leukemic cells, concomitant with a reduction in c-<i>MYC</i> expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors

Validation of the orientation of PlyA.

<p>(A) Proposed orientation of PlyA dimer (pink) and interface with PlyB C-terminal trefoil (green). Trp 6 is shown as purple spheres. (B) Western blot showing PlyA binding to red blood cells when untagged or C-terminally tagged but not when N-terminally tagged.</p