Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.

Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter.

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.

Bacteriophage-encoded lethal membrane disruptors: Advances in understanding and potential applications.

Holins and spanins are bacteriophage-encoded membrane proteins that control bacterial cell lysis in the final stage of the bacteriophage reproductive cycle. Due to their efficient mechanisms for lethal membrane disruption, these proteins are gaining interest in many fields, including the medical, food, biotechnological, and pharmaceutical fields. However, investigating these lethal proteins is challenging due to their toxicity in bacterial expression systems and the resultant low protein yields have hindered their analysis compared to other cell lytic proteins. Therefore, the structural and dynamic properties of holins and spanins in their native environment are not well-understood. In this article we describe recent advances in the classification, purification, and analysis of holin and spanin proteins, which are beginning to overcome the technical barriers to understanding these lethal membrane disrupting proteins, and through this, unlock many potential biotechnological applications.

Evaluation of Biopolymer Materials and Synthesis Techniques to Develop a Rod-Shaped Biopolymer Surrogate for Legionella pneumophila.

Biopolymer microparticles have been developed for applications that require biocompatibility and biodegradability, such as drug delivery. In this study, we assessed the production of microparticles using carnauba wax, κ-carrageenan, alginate, and poly (lactic-co-glycolic acid) (PLGA) with the aim of developing a novel, DNA-tracer-loaded, biopolymer surrogate with a size, shape, surface charge, and relative hydrophobicity similar to stationary-phase Legionella pneumophila to mimic the bacteria's mobility and persistence in engineered water systems. We found that the type and concentration of biopolymer, reaction conditions, and synthesis methods affected the morphology, surface charge, relative hydrophobicity, and DNA tracer loading efficiency of the biopolymer microparticles produced. Carnauba wax, κ-carrageenan, and alginate (Protanal®, and low and medium viscosity) produced highly polydisperse microspheres. In contrast, PLGA and alginate-CaCO3 produced uniform microspheres and rod-shaped microparticles, respectively, with high DNA tracer loading efficiencies (PLGA 70% and alginate-CaCO3 95.2 ± 5.7%) and high reproducibilities. Their synthesis reproducibility was relatively high. The relative hydrophobicity of PLGA microspheres closely matched the cell surface hydrophobicity of L. pneumophila but not the bacterial morphology, whereas the polyelectrolyte layer-by-layer assembly was required to enhance the relative hydrophobicity of alginate-CaCO3 microparticles. Following this surface modification, alginate-CaCO3 microparticles represented the best match to L. pneumophila in size, morphology, surface charge, and relative hydrophobicity. This new biopolymer surrogate has the potential to be used as a mimic to study the mobility and persistence of L. pneumophila in water systems where the use of the pathogen is impractical and unsafe.

Frequency of Hereditary Hemochromatosis Gene (HFE) Variants in Sri Lankan Transfusion-Dependent Beta-Thalassemia Patients and Their Association With the Serum Ferritin Level.

Introduction: Co-inheritance of hereditary hemochromatosis (HFE) gene variants p. C282Y and p.H63D worsen iron overload in transfusion-dependent thalassemia. Data on the HFE gene variants in Sri Lankan patients with thalassemia have not been extensively studied. This study aimed to analyze the p.C282Y and p.H63D variants in transfusion-dependent beta (ß) and HbE/ß-thalassemia patients and establish an association between these variants and their serum ferritin levels. Materials and Methods: A total of 125 transfusion-dependent ß-thalassemia major and HbE/ß thalassemia patients were tested for the c.845G>A (p.C282Y) and c.187C>G (p.H63D) HFE gene variants using the multiplex Amplification Refractory Mutation System Polymerase Chain Reaction method. For phenotype-genotype correlation, serum ferritin levels, the erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels were measured. The standard descriptive statistics were used for data analysis. Results: The study cohort consisted of transfusion-dependent 123 ß-thalassemia and 2 HbE/ß-thalassemia patients. The p.C282Y variant was not detected in any patient; allele frequency for the wild type (c.845GG) was 100%. Twenty-three patients were heterozygous for the p.H63D variant allele, and the allele frequencies were c.187CC 91.8%, c.187CG 9.2%, and c.187GG 0%. The mean serum ferritin level was relatively higher (mean level 4,987 ng/ml) in the p.H63D heterozygous (c.187CG) group compared to the wild type (c.187CC) group (mean level 4,571 ng/ml), but the difference was statistically not significant (p = 0.865). Among the total study population, CRP, ESR, and serum glutamine aspartate transaminase (SGPT) were elevated in 9 (7.2%), 65 (52%), and 82 (65.6%) patients, respectively. Among the p.H63D c.187CG group, elevated CRP, ESR, and SGPT were present in 5 (5%), 15 (12%), and 18 (14.4%) patients, respectively. The detected sample number was low to correlate with the confounding effect of inflammatory disorders and liver damage on the serum ferritin levels. Conclusions: The HFE gene variant p.C282Y is unlikely to cause iron overload in the Asian ß-thalassemia patients; the rarity of this variant in the study cohort replicates the findings of other South Asian population studies of this variant. The presence of the p.H63D variant could be a potential risk factor for iron overload in the ß-thalassemia patients. A more extensive cohort study is required to validate this finding.

The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability.

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.

Water tracking in surface water, groundwater and soils using free and alginate-chitosan encapsulated synthetic DNA tracers.

Investigating contamination pathways and hydraulic connections in complex hydrological systems will benefit greatly from multi-tracer approaches. The use of non-toxic synthetic DNA tracers is promising, because unlimited numbers of tracers, each with a unique DNA identifier, could be used concurrently and detected at extremely low concentrations. This study aimed to develop multiple synthetic DNA tracers as free molecules and encapsulated within microparticles of biocompatible and biodegradable alginate and chitosan, and to validate their field utility in different systems. Experiments encompassing a wide range of conditions and flow rates (19 cm/day-39 km/day) were conducted in a stream, an alluvial gravel aquifer, a fine coastal sand aquifer, and in lysimeters containing undisturbed silt loam over gravels. The DNA tracers were identifiable in all field conditions investigated, and they were directly detectable in the stream at a distance of at least 1 km. The DNA tracers showed promise at tracking fast-flowing water in the stream, gravel aquifer and permeable soils, but were unsatisfactory at tracking slow-moving groundwater in the fine sand aquifer. In the surface water experiments, the microencapsulated DNA tracers' concentrations and mass recoveries were 1-3 orders of magnitude greater than those of the free DNA tracers, because encapsulation protected them from environmental stressors and they were more negatively charged. The opposite was observed in the gravel aquifer, probably due to microparticle filtration by the aquifer media. Although these new DNA tracers showed promise in proof-of-concept field validations, further work is needed before they can be used for large-scale investigations.

On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering.

Bacteriophage endolysins have the potential to be a long-term antibacterial replacement for antibiotics. The exogenous application of endolysins on some bacteria results in rapid cell lysis. The prospects for endolysins are furthered by the ability to engineer them; novel endolysins can be developed with optimised stability, specificity, and lytic function. But the success of endolysin engineering and application requires a comprehensive understanding of the relationship between the enzymes biochemical, biophysical and bacteriolytic properties. Here, we examine their catalytic mechanisms, opportunities for developing novel endolysins, and highlight areas where a better understanding would support their long-term success as antibacterial agents.

The spectrum of deletions and duplications in the dystrophin (DMD) gene in a cohort of patients with Duchenne muscular dystrophy in Sri Lanka.

Background: Duchenne muscular dystrophy (DMD), which affects 1 in 3500 newborn males, is the most common fatal neurodegenerative disorder in children. Deletions and duplications in the DMD gene are the most common underlying etiological factors. Materials and Methods: Fifty consecutive children with DMD were screened for deletions and duplications in the DMD gene using Multiple Ligation-binding Probe Amplification (MLPA). Results: Forty (80%) children had deletions and 4 (8%) had duplications. Single exon involvement was seen in 8 (16%), two exon involvement was seen in 3 (6%), three exon involvement was seen in 6 (12%) children, and four exon involvement in 1 (2%) child. More than four exon involvement were seen in 26 (52%) children. The most common deletion was the deletion spanning from exon 45 to exon 52, which was seen in 6 (12%) children. The next common exon deletion was single exon 45 deletion seen in 4 (8%) children. The most frequent mutant region fell within exons 45 to 55 (52%) followed by within exons 21 to 44 (26%) and exons 1 to 20 (26%). The least common region fell within exons 56 to 79 (4%). Conclusion: The deletion/duplication pattern seen in this cohort of children with DMD was similar to that reported among other global populations.

Autosomal dominant hereditary ataxia in Sri Lanka.

BACKGROUND: Spinocerebellar ataxias (SCA) are a group of hereditary neurodegenerative disorders. Prevalence of SCA subtypes differ worldwide. Autosomal dominant ataxias are the commonest types of inherited ataxias seen in Sri Lanka. The aim of the study is to determine the genetic etiology of patients with autosomal dominant ataxia in Sri Lanka and to describe the clinical features of each genetic subtype. METHODS: Thirty four patients with autosomal dominant ataxia were recruited. For every patient the following was done: recording of clinical details and genotyping for SCA 1, 2, 3, 6, 7, 8, 12, and 17. RESULTS: Sixty one per cent of the subjects were identified as SCA1. One subject had SCA2, 12 remain unidentified. Mean age at onset was 34.8 ± 10years for SCA1 and 32.7 ± 9.8 for non SCA1. 76% of SCA1 patients and 50% of non SCA1 were using walking aids. Quantification of symptoms and signs were similar in the SCA1 and non SCA1 groups. Clinical depression was evidenced in 68.4% of SCA1 and 75% non SCA-1 patients. Mean CAG repeat length in SCA1 patients was 52.0 ± 3.8, with greater anticipation seen with paternal inheritance. CONCLUSION: SCA1 was the predominant subtype and showed similar phenotype to previous reports. However, disease severity was higher and depression more prevalent in this population than previously described.