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Paenibacilluslarvae (American foulbrood) is a lethal and arguably the most destructive and economically important notifiable bacterial disease that severely impacts brood and colonies of the Apis mellifera (Western honey bee) worldwide. Detection in apiaries requires visual inspection of clinically symptomatic hives, which is unreliable, with laboratory confirmation required for definitive diagnosis. These methods can be costly, time-consuming, and require access to specialised equipment operated by experienced personnel. Disease confirmation is essential for notifiable diseases to mitigate spread and economic damages; therefore, rapid, sensitive, and specific point of care diagnostics are critical to prevent misdiagnosis and further outbreaks. To improve diagnostic turnaround, we developed a highly sensitive and specific novel loop-mediated isothermal amplification (LAMP) assay for the detection of P. larvae (AFB-LAMP), designed to amplify a small region of the DNA gyrase subunit B gene (GyrB) with 100% specificity demonstrated against non-target bacterial species of the honey bee gut microbiome and analytical sensitivity of 5 × 10−7 ng P. larvae with detection times within 20 min. To further reduce diagnostic resources and times, a bead-beating DNA extraction method suitable for field use was optimised which resulted in an AFB-LAMP diagnostic sensitivity and specificity of 97 and 98%, respectively. Thus, this AFB-LAMP is applicable for use in the field allowing for improved disease management of an agriculturally important species. [ABSTRACT FROM AUTHOR]

Simple Summary: Bacterial infections continue to cause ongoing health problems in the global equine industry, resulting in substantial loss of valuable economic contributions worldwide. Biosecurity-relevant equine bacterial diseases have resulted in restricted importation and exportation of horses, impediment of breeding, and cancellation of culturally important events. Vigilant, rapid diagnostics and surveillance of such diseases are vital to prevent outbreaks and to provide timely management and treatment strategies. However, current methodologies are outdated and, at times, unreliable. This review outlines how the modern technique of loop-mediated isothermal amplification can aid in the protection of the equine industry, and the current advancements further its ability for in-field deployment. Horses play an important role throughout the world, whether for work, culture, or leisure, providing an ever-growing significant contribution to the economy. The increase in importation and movement of horses, both nationally and internationally, has inevitably allowed for the global equine industry to grow. Subsequently, however, the potential for transmission of fatal equine bacterial diseases has also escalated, and devasting outbreaks continue to occur. To prevent such events, disease surveillance and diagnosis must be heightened throughout the industry. Current common, or "gold-standard" techniques, have shown to be inadequate at times, thus requiring newer technology to impede outbreaks. Loop-mediated isothermal amplification (LAMP) has proven to be a reliable, rapid, and accessible tool in both diagnostics and surveillance. This review will discuss equine bacterial diseases of biosecurity relevance and their current diagnostic approaches, as well as their respective LAMP assay developments. Additionally, we will provide insight regarding newer technology and advancements associated with this technique and their potential use for the outlined diseases. [ABSTRACT FROM AUTHOR]

AB5 toxins are important virulence factors for several major bacterial pathogens, including Bordetella pertussis, Vibrio cholerae, Shigella dysenteriae and at least two distinct pathotypes of Escherichia coli. The AB5 toxins are so named because they comprise a catalytic A-subunit, which is responsible for disruption of essential host functions, and a pentameric B-subunit that binds to specific glycan receptors on the target cell surface. The molecular mechanisms by which the AB5 toxins cause disease have been largely unravelled, including recent insights into a novel AB5 toxin family, subtilase cytotoxin (SubAB). Furthermore, AB5 toxins have become a valuable tool for studying fundamental cellular functions, and are now being investigated for potential applications in the clinical treatment of human diseases. [Copyright &y& Elsevier]

Summary: Ligation of the αβ T cell receptor (TCR) by a specific peptide-loaded major histocompatibility complex (pMHC) molecule initiates T cell signaling via the CD3 complex. However, the initial events that link antigen recognition to T cell signal transduction remain unclear. Here we show, via fluorescence-based experiments and structural analyses, that MHC-restricted antigen recognition by the αβ TCR results in a specific conformational change confined to the A-B loop within the α chain of the constant domain (Cα). The apparent affinity constant of this A-B loop movement mirrored that of αβ TCR-pMHC ligation and was observed in two αβ TCRs with distinct pMHC specificities. The Ag-induced A-B loop conformational change could be inhibited by fixing the juxtapositioning of the constant domains and was shown to be reversible upon pMHC disassociation. Notably, the loop movement within the Cα domain, although specific for an agonist pMHC ligand, was not observed with a pMHC antagonist. Moreover, mutagenesis of residues within the A-B loop impaired T cell signaling in an in vitro system of antigen-specific TCR stimulation. Collectively, our findings provide a basis for the earliest molecular events that underlie Ag-induced T cell triggering. [Copyright &y& Elsevier]

Abstract: Spirochetes of the genus Leptospira cause leptospirosis in humans and animals worldwide. Proteins exposed on the bacterial cell surface are implicated in the pathogenesis of leptospirosis. However, the biological role of the majority of these proteins is unknown; this is principally due to the lack of genetic systems for investigating Leptospira and the absence of any structural information on leptospiral antigens. To address this, we have determined the 2.0-Å-resolution structure of the lipoprotein LipL32, the most abundant outer-membrane and surface protein present exclusively in pathogenic Leptospira species. The extracellular domain of LipL32 revealed a compact, globular, “jelly-roll” fold from which projected an unusual extended β-hairpin that served as a principal mediator of the observed crystallographic dimer. Two acid-rich patches were also identified as potential binding sites for positively charged ligands, such as laminin, to which LipL32 has a propensity to bind. Although LipL32 shared no significant sequence identity to any known protein, it possessed structural homology to the adhesins that bind components of the extracellular matrix, suggesting that LipL32 functions in an analogous manner. Moreover, the structure provides a framework for understanding the immunological role of this major surface lipoprotein. [Copyright &y& Elsevier]

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death. [ABSTRACT FROM AUTHOR]

The waxy cell wall is crucial to the survival of mycobacteria within the infected host. The cell wall is a complex structure rich in unusual molecules that includes two related lipoglycans, the phosphatidylinositol mannosides (PIMs) and lipoarabinomannans (LAMs). Many proteins implicated in the PIM/LAM biosynthetic pathway, while attractive therapeutic targets, are poorly defined. The 2.4Å resolution crystal structure of an essential lipoprotein, LpqW, implicated in LAM biosynthesis is reported here. LpqW adopts a scaffold reminiscent of the distantly related, promiscuous substrate-binding proteins of the ATP-binding cassette import system. Nevertheless, the unique closed conformation of LpqW suggests that mycobacteria and other closely related pathogens have hijacked this scaffold for use in key processes of cell wall biosynthesis. In silico docking provided a plausible model in which the candidate PIM ligand binds within a marked electronegative region located on the surface of LpqW. We suggest that LpqW represents an archetypal lipoprotein that channels intermediates from a pathway for mature PIM production into a pathway for LAM biosynthesis, thus controlling the relative abundance of these two important components of the cell wall. [Copyright &y& Elsevier]

The underlying basis of major histocompatibility complex (MHC) restriction is unclear. Nevertheless, current data suggest that a common thermodynamic signature dictates αβ T cell receptor (TcR) ligation. To evaluate whether this thermodynamic signature defines MHC restriction, we have examined the thermodynamic basis of a highly characterized immunodominant TcR interacting with its cognate peptide-MHC-1 ligand. Surprisingly, we observed this interaction to be governed by favorable enthalpic and entropic forces, which is in contrast to the prevailing generality, namely, enthalpically driven interactions combined with markedly unfavorable entropic forces. We conclude that extrinsic molecular factors, such as coreceptor ligation, conformational adjustments involved in TcR signaling, or constraints dictated by higher-order arrangement of ligated TcRs, might play a greater role in guiding MHC restriction than appreciated previously. [ABSTRACT FROM AUTHOR]

Proteins destined for all submitochondrial compartments are translocated across the outer mitochondrial membrane by the TOM (translocase of the outer membrahe) complex, which consists of a number of specialized receptor subunits that bind mitochondrial precursor proteins for delivery into the translocation channel. One receptor, Tom70, binds large, hydrophobic mitochondrial precursors. The current model of Tom70-mediated import involves multiple dimers of the receptor recognizing a single molecule of substrate. Here we show via a battery of biophysical and spectroscopic techniques that the cytosolic domain of Tom70 is an elongated monomer. Thermal and urea-induced denaturation revealed that the receptor, which unfolds via a multistate pathway, is a relatively unstable molecule undergoing major conformational change at physiological temperatures. The data suggest that the malleability of the monomeric Tom70 receptor is an important factor in mitochondrial import. [ABSTRACT FROM AUTHOR]

Thousands of polypeptides with diverse biochemical properties, some of which are extremely hydrophobic, are targeted from cytoplasmic ribosomes to the surface of mitochondria. Localised synthesis, as well as transient interactions with a wide array of molecular chaperones and other cytoplasmic factors, can promote productive interaction of mitochondrial proteins with the TOM complex to initiate protein import into mitochondria. [Copyright &y& Elsevier]

Simple Summary: Equine viral diseases remain a prominent concern for human and equine health globally. Many of these viruses are of primary biosecurity concern to countries that import equines where these viruses are not present. In addition, several equine viruses are zoonotic, which can have a significant impact on human health. Current diagnostic techniques are both time consuming and laboratory-based. The ability to accurately detect diseases will lead to better management, treatment strategies, and health outcomes. This review outlines the current modern isothermal techniques for diagnostics, such as loop-mediated isothermal amplification and insulated isothermal polymerase chain reaction, and their application as point-of-care diagnostics for the equine industry. The global equine industry provides significant economic contributions worldwide, producing approximately USD $300 billion annually. However, with the continuous national and international movement and importation of horses, there is an ongoing threat of a viral outbreak causing large epidemics and subsequent significant economic losses. Additionally, horses serve as a host for several zoonotic diseases that could cause significant human health problems. The ability to rapidly diagnose equine viral diseases early could lead to better management, treatment, and biosecurity strategies. Current serological and molecular methods cannot be field-deployable and are not suitable for resource-poor laboratories due to the requirement of expensive equipment and trained personnel. Recently, isothermal nucleic acid amplification technologies, such as loop-mediated isothermal amplification (LAMP) and insulated isothermal polymerase chain reaction (iiPCR), have been developed to be utilized in-field, and provide rapid results within an hour. We will review current isothermal diagnostic techniques available to diagnose equine viruses of biosecurity and zoonotic concern and provide insight into their potential for in-field deployment. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is a renewed cause of devastation in the developing world. Critical to the success of this re-emerging pathogen is its unusual waxy cell wall, which is rich in rare components including lipoarabinomannan (LAM) and its precursors, the phosphatidylinositol mannosides (PIMs). Balanced synthesis of these related glycolipids is intrinsic to both cell-wall integrity and virulence in M. tuberculosis and presents a promising, albeit poorly defined, therapeutic target. Here, the expression, purification and crystallization of an essential 600-­amino-acid lipoprotein, LpqW, implicated in this process are reported. Crystals of LpqW were grown using 20–24%( w/ v) PEG 4000, 8–­16%( v/ v) 2-­propanol, 100 m M sodium citrate pH 5.5 and 10 m M DTT. A complete data set was collected at 2.4 Å using synchrotron radiation on a crystal belonging to space group C222, with unit-cell parameters a = 188.57, b = 312.04, c = 104.15 Å. Structure determination is under way. [ABSTRACT FROM AUTHOR]

Reef-building corals contain fluorescent pigments termed pocilloporins that function by regulating the light environment of coral and acting as a photoprotectant in excessive sunlight. These pocilloporins are related to the monomeric green fluorescent protein and the tetrameric DsRed fluorescent proteins, which have widespread use as biotechnological tools. An intensely blue-coloured pocilloporin, termed Rtms5, was expressed in Escherichia coli, purified and crystallized. Rtms5 was shown to be tetrameric, with deep blue crystals that diffract to 2.2 Å resolution and belong to space group I4[sub 1]22. The colour of this pocilloporin was observed to be sensitive to pH and a yellow (pH 3.5) and a red form (pH 4.5) of Rtms5 were also crystallized. These crystals belong to space group P4[sub 2]22 and diffract to 2.4 Å resolution or better. [ABSTRACT FROM AUTHOR]

The sheep body louse (Bovicola ovis) commonly referred to as sheep lice are small chewing ectoparasites of sheep. Infection results in significant economic costs to the Australian sheep industry due to reduced wool quality caused by chronic itching from sheep rubbing and biting fleece. Treatment relies on use of insecticides; however, resistance has developed against pyrethroid and other insect growth regulator lousicides. There is urgent need to develop cost-effective lice management to reduce the use of insecticides, with the application of insecticidal treatments only applied when an infestation is detected. However, the current detection method relies on fleece parting for detection of B. ovis which is highly dependent on the skill of the inspector, the number of sheep examined, and the prevalence and severity of the infestation. To improve B. ovis detection, a highly sensitive (5 × 10−8 ng/μL) and specific multiplex quantitative PCR which simultaneously detects sheep lice and sheep DNA was developed. In addition, a B. ovis loop-mediated isothermal amplification (LAMP) assay was developed for field use. The B. ovis LAMP (Bov-LAMP) assay was optimized to reliably detect B. ovis from wool samples down to 5 × 10−6 ng/μL, with time to positive (Tp) < 10 min. Both assays demonstrate high sensitivity and specificity, enabling rapid identification of B. ovis DNA from sheep fleece samples and have the capacity to be used for ongoing management and surveillance of B. ovis in Australian sheep flocks. [ABSTRACT FROM AUTHOR]

The article discusses the study of Call and colleagues, which provides the structural basis for the assembly of immunoreceptor natural killer group 2C (NKG2C) with signaling adaptor molecule DAP12. The study used mutagenesis and in vitro assembly assays and recombinant linked trimer. Moreover, the study reveals that the loss of threonine caused impaired assembly of DAP12 and NKG2C and that NKG2C transmembrane (TM) helix was packed against the lateral surface of DAP12 homodimer.

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of l-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and l-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues. [ABSTRACT FROM AUTHOR]

Triclabendazole (TCBZ) is the only chemical that kills early immature and adult Fasciola hepatica (liver fluke) but widespread resistance to the drug greatly compromises fluke control in livestock and humans. The mode of action of TCBZ and mechanism(s) underlying parasite resistance to the drug are not known. Due to the high prevalence of TCBZ resistance (TCBZ-R), effective management of drug resistance is now critical for sustainable livestock production. Here, we discuss the current status of TCBZ-R in F. hepatica , the global distribution of resistance observed in livestock, the possible mechanism(s) of drug action, the proposed mechanisms and genetic basis of resistance, and the prospects for future control of liver fluke infections using an integrated parasite management (IPM) approach. [ABSTRACT FROM AUTHOR]

Vertebrate sialic acids (Sias) display much diversity in modifications, linkages, and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan–protein interactions. A microarray presenting ~150 structurally defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization, and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the nonreducing end, with 3 digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveal >113,000 likely linear sialyltrisaccharides in nature. Notably, a biantennary N -glycan with 2 terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N -glycan with 3 terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4- O -acetylated-Sias not recognized by a Yersinia pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), Salmonella Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB). [ABSTRACT FROM AUTHOR]

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors. [ABSTRACT FROM AUTHOR]

Summary: The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 Å resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain. [Copyright &y& Elsevier]

The energetic bases of T cell recognition are unclear. Here, we studied the 'energetic landscape' of peptide-major histocompatibility complex (pMHC) recognition by an immunodominantaßT cell receptor (TCR). We quantified and evaluated the effect of natural and systematic substitutions in the complementarity-determining region (CDR) loops on ligand binding in the context of the structural detail of each component of the immunodominant TCR-pMHC complex. The CDR1 and CDR2 loops contributed minimal energy through direct recognition of the antigen and instead had a chief function in stabilizing the ligated CDR3 loops. The underlying energetic basis for recognition lay in the CDR3 loops. Therefore the energetic burden of the CDR loops in the TCR-pMHC interaction is variable among TCRs, reflecting the inherent adaptability of the TCR in ligating different ligands. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB5 toxin, inhibits Gαi/o G proteins and has proven useful for interrogating inhibitory G protein signalling. Pertussis toxin, however, does not inhibit one member of the inhibitory G protein family, Gαz. The role of Gαz signalling has been neglected largely due to a lack of inhibitors. Recently, the identification of another Pertussis-like AB5 toxin was described. Here we show that this toxin, that we call OZITX, specifically inhibits Gαi/o and Gαz G proteins and that expression of the catalytic S1 subunit is sufficient for this inhibition. We identify mutations that render Gα subunits insensitive to the toxin that, in combination with the toxin, can be used to interrogate the signalling of each inhibitory Gα G protein. A recently identified pertussis toxin-like AB5 toxin, OZITX, is found to inhibit Gαi/o and Gαz G proteins. In combination with directed mutations, it is a useful tool for interrogating Gαi/o/z G protein subunits individually. [ABSTRACT FROM AUTHOR]

Molluscs are major contributors to the international and Australian aquaculture industries, however, their immune systems remain poorly understood due to limited access to draft genomes and evidence of divergences from model organisms. As invertebrates, molluscs lack adaptive immune systems or 'memory', and rely solely on innate immunity for antimicrobial defence. Hemolymph, the circulatory fluid of invertebrates, contains hemocytes which secrete effector molecules with immune regulatory functions. Interactions between mollusc effector molecules and bacterial and fungal pathogens have been well documented, however, there is limited knowledge of their roles against viruses, which cause high mortality and significant production losses in these species. Of the major effector molecules, only the direct acting protein dicer-2 and the antimicrobial peptides (AMPs) hemocyanin and myticin-C have shown antiviral activity. A better understanding of these effector molecules may allow for the manipulation of mollusc proteomes to enhance antiviral and overall antimicrobial defence to prevent future outbreaks and minimize economic outbreaks. Moreover, effector molecule research may yield the description and production of novel antimicrobial treatments for a broad host range of animal species. [ABSTRACT FROM AUTHOR]

We have crystallized and subsequently determined to 2.0-Å resolution the crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. The structure of the protomer, which adopts a β-can topology, is similar to that of the related monomeric green fluorescent protein (GFP). The quaternary structure of eqFP611, a tetramer exhibiting 222 symmetry, is similar to that observed for the more closely related red fluorescent protein DsRed and the chromoprotein Rtms5. The unique chromophore sequence (Met[sup 63]-Tyr[sup 64]-Gly[sup 65]) of eqFP611, adopts a coplanar and trans conformation within the interior of the β-can fold. Accordingly, the eqFP611 chromophore adopts a significantly different conformation in comparison to the chromophore conformation observed in GFP, DsRed, and Rtms5. The coplanar chromophore conformation and its immediate environment provide a structural basis for the far red, highly fluorescent nature of eqFP611. The eqFP611 structure extends our knowledge on the range of conformations a chromophore can adopt within closely related members of the green fluorescent protein family. [ABSTRACT FROM AUTHOR]

Examines the crystal structure of myelin oligodendrocyte glycoprotein, a multiple sclerosis autoantigen. Expression, refolding and crystallization trials on protein; Automated structure comparisons; Dimeric species of the myelin oligodendrocyte glycoprotein.

Reef-building corals contain host pigments, termed pocilloporins, that function to regulate the light environment of their resident microalgae by acting as a photoprotectant in excessive sunlight. We have determined the crystal structure of an intensely blue, nonfluorescent pocilloporin to 2.2 A˚ resolution and a genetically engineered fluorescent variant to 2.4 A˚ resolution. The pocilloporin chromophore structure adopts a markedly different conformation in comparison with the DsRed chromophore, despite the chromophore sequences (Gln-Tyr-Gly) being identical; the tyrosine ring of the pocilloporin chromophore is noncoplanar and in the trans configuration. Furthermore, the fluorescent variant adopted a noncoplanar chromophore conformation. The data presented here demonstrates that the conformation of the chromophore is highly dependent on its immediate environment. [Copyright &y& Elsevier]

The nascent polypeptide-associated complex (NAC) is a peripheral component of cytoplasmic ribosomes, and interacts with nascent chains as they leave the ribosome. Yeast mutants lacking NAC translate polypeptides normally, but have fewer ribosomes associated with the mitochondrial surface. The mutants lacking NAC suffer mitochondrial defects and have decreased levels of proteins like fumarase, normally targeted to mitochondria co-translationally. NAC might contribute to a ribosomal environment in which amino-terminal, mitochondrial targeting sequences can effectively adopt their appropriate conformation. [Copyright &y& Elsevier]

Examines the effects of mitochondrial protein targeting suppression on the cytoplasmic polysomes translation. Synthesis of messenger RNA in the nucleus of eukaryotic organisms; Factors affecting the mitochondrial protein targeting; Importation of mitochondrial precursor protein into the mitochondria.

Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a 'rigorous' FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a 'rigorous' FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture. • On-farm applicable DNA extraction method was developed for fasciolosis. • Loop-mediated isothermal amplification method (LAMP) was optimised for fasciolosis. • LAMP results were compared to faecal egg counts and polymerase chain reaction. • High sensitivity and specificity of the LAMP method in the laboratory. • High sensitivity and low specificity of the LAMP method when applied on-farm. [ABSTRACT FROM AUTHOR]

Simple Summary: Retaining sustainable agricultural systems is essential to feed the expanding population. Helminth parasite infections impact livestock production values and yields, including infections in goats, which are often overlooked. There is a requirement to collate caprine-specific knowledge surrounding fasciolosis, caused by Fasciola hepatica and Fasciola gigantica (liver flukes). Current liver fluke control methods include drug application and pasture management. This review aims to outline goat-specific fasciolosis vaccine development and the potential for alternative control methods. The disease fasciolosis is caused by the liver flukes Fasciola hepatica and F. gigantica, which infect a wide range of mammals and production livestock, including goats. These flatworm parasites are globally distributed and predicted to cost the livestock industry a now conservative USD 3 billion per year in treatment and lowered on-farm productivity. Infection poses a risk to animal welfare and results in lowered fertility rates and reduced production yields of meat, milk and wool. This zoonotic disease is estimated to infect over 600 million animals and up to 2.4 million humans. Current and future control is threatened with the global emergence of flukes resistant to anthelmintics. Drug resistance calls for immediate on-farm parasite management to ensure treatments are effective and re-infection rates are kept low, while a sustainable long-term control method, such as a vaccine, is being developed. Despite the recent expansion of the goat industry, particularly in developing countries, there are limited studies on goat-focused vaccine control studies and the effectiveness of drug treatments. There is a requirement to collate caprine-specific fasciolosis knowledge. This review will present the current status of liver fluke caprine infections and potential control methods for application in goat farming. [ABSTRACT FROM AUTHOR]

Objective: Infectious laryngotracheitis (ILT) is an acute and highly contagious viral respiratory disease of poultry, caused by gallid herpesvirus 1 (ILTV), which causes significant economic losses. Due to recent outbreaks of ILT in Australia, it has been proposed that ILT could be transmitted between poultry sheds by airborne transmission; however, there has never been direct detection of ILTV from air samples. We aimed to optimize a sampling system for the detection of airborne ILTV in poultry sheds. Results: Poultry farms with a known outbreaks of ILT were used for detection of airborne ILTV. Infected chickens were verified by detection of ILTV nucleic acid in feather shafts with all farms being positive. Using a liquid cyclonic impinging device, it was found that recovery and detection of airborne ILTV was possible in alkaline PEG buffer. Additional sampling was performed at different heights to determine the presence of ILTV in the air. In farm 3, all three air samples at both heights were positive for ILTV while at farm 2 only one sample at 45 cm was positive. We envisaged in the future air sampling will be able to detect and track potential transmission of ILTV both inside and outside of the poultry shed. [ABSTRACT FROM AUTHOR]

• Ruminant-specific galectins 11 and 14 are upregulated upon parasitic infection. • Galectin 11 and 14 ligands were identified in Fasciola hepatica. • Galectin 14 interacts with a diverse range of 255 F. hepatica glycoproteins. • Galectin 11 weakly interacts with glycoproteins of F. hepatica. • This method provides information about the host-parasite interface and potential vaccine candidates. Fasciola hepatica is a globally distributed zoonotic trematode that causes fasciolosis in livestock, wildlife, ruminants and humans. Fasciolosis causes a significant economic impact on the agricultural sector and affects human health. Due to the increasing prevalence of triclabendazole resistance in F. hepatica , alternative treatment methods are required. Many protein antigens have been trialled as vaccine candidates with low success, however, the tegument of F. hepatica is highly glycosylated and the parasite-derived glycoconjugate molecules have been identified as an important mediator in host-parasite interactions and as prime targets for the host immune system. Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are two ruminant-specific glycan-binding proteins, showing upregulation in the bile duct of sheep infected with F. hepatica , which are believed to mediate host-parasite interaction and innate immunity against internal parasites. For the first known time, this study presents the ligand profile of whole worm and tegument extracts of F. hepatica that interacted with immobilised LGALS-11 and LGALS-14. LGALS-14 interacted with a total of 255 F. hepatica proteins. The protein which had the greatest interaction was identified as an uncharacterised protein which contained a C-type lectin domain. Many of the other proteins identified were previously trialled vaccine candidates including glutathione S-transferase, paramyosin, cathepsin L, cathepsin B, fatty acid binding protein and leucine aminopeptidase. In comparison to LGALS-14, LGALS-11 interacted with only 49 F. hepatica proteins and it appears to have a much smaller number of binding partners in F. hepatica. This is, to our knowledge, the first time host-specific lectins have been used for the enrichment of F. hepatica glycoproteins and this study has identified a number of glycoproteins that play critical roles in host-parasite interactions which have the potential to be novel vaccine candidates. [ABSTRACT FROM AUTHOR]

Dichelobacter nododus is the causative agent of footrot, a major disease of sheep that creates welfare concerns and large economic loss. The virulence of D. nododus depends on the presence of extracellular proteases, AprV2 and AprB2, which differ by one amino acid. Strains possessing AprV2 can cause clinically virulent disease, while AprB2 may cause clinically benign disease. Current methods for detecting D. nodosus are difficult, laborious and time consuming. New techniques capable of rapidly detecting and typing D. nodosus are needed to aid control programs. Molecular methods, like real-time polymerase chain reaction (rtPCR) can detect aprV2 and aprB2, however, this assay is not field-deployable and cannot support local decision-making during an outbreak. Here we present a field-based molecular assay for detecting aprV2, using loop mediated isothermal amplification (LAMP). The aprV2 LAMP (VDN LAMP) assay was optimised to reliably detect aprV2 from laboratory purified genomic (gDNA) of virulent D. nodosus down to 5x10-3 ng μL-1, with time to positive (Tp) ≤ 16 minutes, while aprB2 was unreliably detected at 5 ng μL-1 from 16–20 minutes. The use of field collected samples that were rtPCR positive for aprB2 resulted in no amplification, while aprV2 positive field samples by VDN LAMP assay are defined as having Tps’ of < 20 minutes and melting temperature between 88.0–88.9°C. When compared to rtPCR, the VDN LAMP was shown to have a diagnostic specificity of 100% and sensitivity of 83.33%. As proof of concept, the VDN LAMP was taken on farm, with all processing occurring in-field. The on farm VDN LAMP successfully detected 91.67% aprV2 positive samples, no aprB2 positive samples (n = 9) or D. nodosus negative (n = 23) samples, with a kappa agreement of ‘almost perfect’ to rtPCR. This highlights the potential of the assay to inform local treatment decisions for management. [ABSTRACT FROM AUTHOR]

Liver fluke ( Fasciola hepatica ) infection is an increasing threat to livestock production resulting in serious economic losses to the beef, dairy and sheep industries in Australia and globally. Triclabendazole (TCBZ) is the main drug used to control liver fluke infections in Australia and the widespread emergence of TCBZ resistance in cattle and sheep threatens liver fluke control. Alternative control measures to lower exposure of livestock to fluke infection would be useful to help preserve the usefulness of current chemical flukicides. Environmental DNA (eDNA) sampling methodology and associated molecular techniques are suited to rapidly assess the presence of pathogens on farms. In the present study, we developed a water sampling method in combination with a multiplex quantitative PCR assay to detect and quantify DNA of F. hepatica and Austropeplea tomentosa ( A. tomentosa ), a crucial intermediate snail host for liver fluke transmission in South-east Australia. The multiplex qPCR assay allows for the independent detection of F. hepatica and A. tomentosa DNA using specific primers and a probe targeting the ITS-2 region of the liver fluke or snail. The method allows the highly specific and sensitive (minimal DNA detection levels to 14–50 fg) detection of F. hepatica or A. tomentosa . The method allows the detection of both liver fluke and snail eDNA in water samples. The effective quantification of liver fluke and snail eDNA in water samples using this assay could potentially allow researchers to both identify and monitor F. hepatica transmission zones on farming properties in South-east Australia which will better inform control strategies, with potential application of the assay worldwide. [ABSTRACT FROM AUTHOR]

Objective: Dichelobacter nodosus is the causative agent of footrot in sheep. Ovine footrot is a major problem in Australia that results in large economic losses and a represents a very significant animal welfare issue. D. nodosus is divided into 10 serogroups (A-I, M), based on sequence variation in the type IV fimbriae gene, fimA. Control of the bacteria is possible through use of serogroup-specific vaccination, however traditional identification of the serogroups of D. nodosus on infected sheep is time-consuming and costly. With the aim of reducing time and cost, a PCR assay was used to identify serogroups of D. nodosus directly from foot swabs of infected sheep in Victoria. Results: It was shown that serogroup B was most common (10 locations), followed by A, G and H (4 locations), I and C (2 locations), D, E and F (1 location). Infections with multiple serotypes were observed in 50% of farms, with the remaining 50% having only a single serogroup detected. The ability to identify serogroups quickly and cheaply direct from foot swabs will aid the understanding of the epidemiology of D. nodosus and support control programs. [ABSTRACT FROM AUTHOR]

αβ T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8+Tcell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8+ T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of aTRBV9+TCRin complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9+TCRadopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14+ TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells. [ABSTRACT FROM AUTHOR]

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+ -binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis- like toxins, and we also identified differences in host target specificity. [ABSTRACT FROM AUTHOR]

A more thorough understanding of the immunological interactions between Fasciola spp. and their hosts is required if we are to develop new immunotherapies to control fasciolosis. Deeper knowledge of the antigens that are the target of the acquired immune responses of definitive hosts against both Fasciola hepatica and Fasciola gigantica will potentially identify candidate vaccine antigens. Indonesian Thin Tail sheep express a high level of acquired immunity to infection by F. gigantica within 4 weeks of infection and antibodies in Indonesian Thin Tail sera can promote antibody-dependent cell-mediated cytotoxicity against the surface tegument of juvenile F. gigantica in vitro. Given the high protein sequence similarity between F. hepatica and F. gigantica , we hypothesised that antibody from F. gigantica -infected sheep could be used to identify the orthologous proteins in the tegument of F. hepatica . Purified IgG from the sera of F. gigantica -infected Indonesian Thin Tail sheep collected pre-infection and 4 weeks p.i. were incubated with live adult F. hepatica ex vivo and the immunosloughate (immunoprecipitate) formed was isolated and analysed via liquid chromatography-electrospray ionisation-tandem mass spectrometry to identify proteins involved in the immune response. A total of 38 proteins were identified at a significantly higher abundance in the immunosloughate using week 4 IgG, including eight predicted membrane proteins, 20 secreted proteins, nine proteins predicted to be associated with either the lysosomes, the cytoplasm or the cytoskeleton and one protein with an unknown cellular localization. Three of the membrane proteins are transporters including a multidrug resistance protein, an amino acid permease and a glucose transporter. Interestingly, a total of 21 of the 38 proteins matched with proteins recently reported to be associated with the proposed small exosome-like extracellular vesicles of adult F. hepatica , suggesting that the Indonesian Thin Tail week 4 IgG is either recognising individual proteins released from extracellular vesicles or is immunoprecipitating intact exosome-like extracellular vesicles. Five extracellular vesicle membrane proteins were identified including two proteins predicted to be associated with vesicle transport/ exocytosis (VPS4, vacuolar protein sorting-associated protein 4b and the Niemann-Pick C1 protein). RNAseq analysis of the developmental transcription of the 38 immunosloughate proteins showed that the sequences are expressed over a wide abundance range with 21/38 transcripts expressed at a relatively high level from metacercariae to the adult life cycle stage. A notable feature of the immunosloughates was the absence of cytosolic proteins which have been reported to be secreted markers for damage to adult flukes incubated in vitro, suggesting that the proteins observed are not inadvertent contaminants leaking from damaged flukes ex vivo. The identification of tegument protein antigens shared between F. gigantica and F. hepatica is beneficial in terms of the possible development of a dual purpose vaccine effective against both fluke species. [ABSTRACT FROM AUTHOR]

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth. [ABSTRACT FROM AUTHOR]

Cannabis sativa L. has been known for at least 2000 years as a source of important, medically significant specialised metabolites and several bio-active molecules have been enriched from multiple chemotypes. However, due to the many levels of complexity in both the commercial cultivation of cannabis and extraction of its specialised metabolites, several heterologous production approaches are being pursued in parallel. In this review, we outline the recent achievements in engineering strategies used for heterologous production of cannabinoids, terpenes and flavonoids along with their strength and weakness. We provide an overview of the specialised metabolism pathway in C. sativa and a comprehensive list of the specialised metabolites produced along with their medicinal significance. We highlight cannabinoid-like molecules produced by other species. We discuss the key biosynthetic enzymes and their heterologous production using various hosts such as microbial and eukaryotic systems. A brief discussion on complementary production strategies using co-culturing and cell-free systems is described. Various approaches to optimise specialised metabolite production through co-expression, enzyme engineering and pathway engineering are discussed. We derive insights from recent advances in metabolic engineering of hosts with improved precursor supply and suggest their application for the production of C. sativa speciality metabolites. We present a collation of non-conventional hosts with speciality traits that can improve the feasibility of commercial heterologous production of cannabis-based specialised metabolites. We provide a perspective of emerging research in synthetic biology, allied analytical techniques and plant heterologous platforms as focus areas for heterologous production of cannabis specialised metabolites in the future. [Display omitted] • Overview of biosynthetic pathway and enzyme attributes of specialised metabolites in C.sativa. • Co-culturing and cell-free systems examined as complementary production strategies. • Enzyme engineering and co-expression of helper proteins discussed as optimisation approaches. • Metabolic engineering strategies to generate hosts with improved precursor supply. • Non-conventional microbes with specialty traits presented as emerging hosts. [ABSTRACT FROM AUTHOR]

Galectins are an evolutionarily conserved family of proteins that translate glycan recognition into cellular effects. Galectin-11 is a unique member of the galectin family that is only expressed in ruminants such as sheep, goat and cattle and that plays a critical role in several important biological processes, such as reproduction and parasite-mediated innate immune responses. Currently, these two areas are of major importance for the sustainability of ruminant livestock production. Despite the emerging biological significance of galectin-11, no structural information is available. It is expected that structural studies will unravel the functional mechanisms of galectin-11 activity. Here, the expression, purification and crystallization of the ruminant-specific galectin-11 from domestic sheep and the collection of X-ray data to 2.0 Å resolution are reported. [ABSTRACT FROM AUTHOR]

The killer cell Ig-like receptor 3DL1 (KIR3DL1) inhibits activation of NK cells upon interaction with HLA class I molecules such as HLA-B*57:01, which contains the Bw4 epitope spanning residues 77-83 (e.g., NLRIALR), and not with HLA allomorphs that possess the Bw6 motif (e.g., HLA-B*08:01), which differ at residues 77, 80, 81, 82, and 83. Although Bw4 residues Ile80 and Arg83 directly interact with KIR3DL1*001, their precise role in determining KIR3DL1 -HLA-Bw4 specificity remains unclear. Recognition of HLA-B*57:01 by either KIR3DL1+ NK cells or the NK cell line YTS transfected with KIR3DL1*001 was impaired by mutation of residues 80 and 83 of HLA-B*57:01 to the corresponding amino acids within the Bw6 motif. Conversely, the simultaneous introduction of three Bw4 residues at positions 80, 82, and 83 into HLA-B*08:01 conferred an interaction with KIR3DI,1*001. Structural analysis of HLA-B*57:01, HLA-B*08:01, and mutants of each bearing substitutions at positions 80 and 83 revealed that Ile80 and Arg83 within the Bw4 motif constrain the conformation of Glu76, primarily through a salt bridge between Arg83 and Glu76. This salt bridge was absent in HLA-Bw6 molecules as well as position 83 mutants of HLA-B*57:01. Mutation of the Bw4 residue Ile80 also disrupted this salt bridge, providing further insight into the role that position 80 plays in mediating KIR3DL1 recognition. Thus, the strict conformation of HLA-Bw4 allotypes, held in place by the Glu76-Arg83 interaction, facilitates KIR3DL1 binding, whereas Bw6 allotypes present a platform on the ⓫ helix that is less permissive for KIR3DL1 binding. [ABSTRACT FROM AUTHOR]

The success of pathogenic mycobacterial species is owing in part to their ability to parasitize the generally inhospitable phagosomal environment of host macrophages, utilizing a variety of strategies to avoid their antimycobacterial capabilities and thereby enabling their survival. A recently identified gene target in Mycobacterium smegmatis, highly conserved within Mycobacterium spp. and denoted MSMEG_5817, has been found to be important for bacterial survival within host macrophages. To gain insight into its function, the crystal structure of MSMEG_5817 has been solved to 2.40 Å resolution. The structure reveals a high level of structural homology to the sterol carrier protein (SCP) family, suggesting a potential role of MSMEG_5817 in the binding and transportation of biologically relevant lipids required for bacterial survival. The lipid-binding capacity of MSMEG_5817 was confirmed by ELISA, revealing binding to a number of phospholipids with varying binding specificities compared with Homo sapiens SCP. A potential lipid-binding site was probed by alanine-scanning mutagenesis, revealing structurally relevant residues and a binding mechanism potentially differing from that of the SCPs. [ABSTRACT FROM AUTHOR]

The present study focussed on investigating CD59-like molecules of Fasciola hepatica. A cDNA encoding a CD59-like protein (termed FhCD59-1) identified previously in the membrane fraction of the F. hepatica tegument was isolated. This homologue was shown to encode a predicted open reading frame (ORF) of 122 amino acids (aa) orthologous to human CD59 with a 25 aa signal peptide, a mature protein containing 10 cysteines and a conserved CD59/Ly-6 family motif "CCXXXXCN". An analysis of cDNAs from two different adult specimens of F. hepatica revealed seven variable types of FhCD59-1 sequences, designated FhCD59-1.1 to FhCD59-1.7, which had 94.3-99.7% amino acid sequence identity upon pairwise comparison. Molecular modeling of FhCD59-1.1 with human CD59 confirmed the presence of the three-finger protein domain found in the CD59 family and predicted three disulphide bonds in the F. hepatica sequence. The interrogation of F. hepatica databases identified two additional sequences, designated FhCD59-2 and FhCD59-3, which had only 23.4-29.5% amino acid identity to FhCD59-1.1. Orthologues of the inferred CD59 protein sequences of F. hepatica were also identified in other flatworms, including Fasciola gigantica, Fascioloides magna, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Clonorchis sinensis, Opisthorchis viverrini, Taenia solium, Echinococcus granulosus and the free living Schmidtea mediterannea. The results revealed a considerable degree of sequence complexity in the CD59-like sequence families in F. hepatica and flatworms. Phylogenetic analysis of CD59-like aa sequences from F. hepatica and flatworms showed that FhCD59-2 clustered with the known surface-associated protein SmCD59-2 of S. mansoni. Relatively well-supported clades specific to schistosomes, fasciolids and opisthorchiids were identified. The qPCR analysis of gene transcription showed that the relative expression of these 3 FhCD59-like sequences varied by 11-47-fold during fluke maturation, from the newly excysted juvenile (NEJ) to the adult stage. These findings suggest that different FhCD59-like sequences play distinct roles during the development of F. hepatica. [ABSTRACT FROM AUTHOR]

Legionella pneumophila is an opportunistic pathogen that replicates within alveolar macrophages resulting in the onset of severe atypical pneumonia. Previously we have identified Lpg1905, a eukaryotic-type ecto-NTPDase (nucleoside triphosphate diphosphohydrolase) from L. pneumophila that was required for optimal intracellular replication and virulence in a mouse lung infection model. In the present study, we characterized the activity of a second eukaryotic-type NTPDase, Lpg0971, from L. pneumophila. We observed that recombinant Lpg0971 hydrolysed only ATP and exhibited divalent cation preference for manganese (II) ions. Similar to lpg1905, an lpg0971 mutant carrying the plasmid pMIP was attenuated in a mouse lung infection model and impaired for replication in human macrophages and amoebae. Increased trafficking of the LCV (Legionella-containing vacuole) to a LAMP-1 (lysosome-associated membrane protein-1)-positive compartment was observed for both the lpg1905 and lpg0971 mutants carrying pMIP. Complementation with either lpg1905 or lpg0971 restored intracellular replication, suggesting that a minimum level of ATPase activity was required for this function. A double lpg1905/0971 mutant was not more impaired for intracellular replication than the single mutants and complementation of the double mutant with lpg0971, but not lpg1905, restored intracellular replication. This suggested that although the NTPDases have overlapping activities they have distinct functions. Unlike many eukaryotic-type proteins from L. pneumophila, neither Lpg1905 nor Lpg0971 were translocated into the host cell by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IV secretion system. Overall our data suggest that the ability of L. pneumophila to replicate in eukaryotic cells relies in part on the ability of the pathogen to hydrolyse ATP within an intracellular compartment. [ABSTRACT FROM AUTHOR]

Aldo-keto reductases (AKR) are a large superfamily of NADPH-dependent oxidoreductases and play a role in detoxification of toxic metabolites. Rv2971, an AKR in Mycobacterium tuberculosis, has recently been identified as a target of isoniazid, a key first-line drug against tuberculosis. Here, the cloning, expression, purification, crystallization and structural characterization of Rv2971 are described. To gain insight into its function, the crystal structure of Rv2971 was successfully determined to 1.60 Å resolution in its unliganded form. The structure exhibits a TIM-barrel fold typical of AKRs, revealing structural characteristics essential for function and substrate specificities, allowing a structural comparison between Rv2971 and other mycobacterial AKRs. [ABSTRACT FROM AUTHOR]

Summary: AB5 toxins are composed of an enzymatic A subunit that disrupts cellular function associated with a pentameric B subunit required for host cell invasion. EcxAB is an AB5 toxin isolated from clinical strains of Escherichia coli classified as part of the cholera family due to B subunit homology. Cholera-group toxins have catalytic ADP-ribosyltransferases as their A subunits, so it was surprising that EcxA did not. We confirmed that EcxAB self-associates as a functional toxin and obtained its structure. EcxAB is a prototypical member of a hybrid AB5 toxin family containing metzincin-type metalloproteases as their active A subunit paired to a cholera-like B subunit. Furthermore, EcxA is distinct from previously characterized proteases and thus founds an AB5-associated metzincin family that we term the toxilysins. EcxAB provides the first observation of conserved B subunit usage across different AB5 toxin families and provides evidence that the intersubunit interface of these toxins is far more permissive than previously supposed. [Copyright &y& Elsevier]

The T cell repertoire comprises αβ and γδ T cell lineages. Although it is established how αβ T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for γδ TCRs. We describe a population of human Vδ1+ γδ T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a γδ TCR binds CD1d-α-galactosylceramide (α-GalCer). The γδ TCR docked orthogonally, over the A′ pocket of CD1d, in which the Vδ1-chain, and in particular the germ line-encoded CDR1δ loop, dominated interactions with CD1d. The TCR γ-chain sat peripherally to the interface, with the CDR3γ loop representing the principal determinant for α-GalCer specificity. Accordingly, we provide insight into how a γδ TCR binds specifically to a lipid-loaded antigen-presenting molecule. [ABSTRACT FROM AUTHOR]