Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria

Invasion of host cells by the malaria pathogen Plasmodium relies on parasite transmembrane adhesins that engage host-cell receptors. Adhesins must be released by cleavage before the parasite can enter the cell, but the processing enzymes have remained elusive. Recent work indicates that the Toxoplasma rhomboid intramembrane protease TgROM5 catalyzes this essential cleavage. However, Plasmodium does not encode a direct TgROM5 homolog. We examined processing of the 14 Plasmodium falciparum adhesins currently thought to be involved in invasion by both model and Plasmodium rhomboid proteases in a heterologous assay. While most adhesins contain aromatic transmembrane residues and could not be cleaved by nonparasite rhomboid proteins, including Drosophila Rhomboid-1, Plasmodium falciparum rhomboid protein (PfROM)4 (PFE0340c) was able to process these adhesins efficiently and displayed novel substrate specificity. Conversely, PfROM1 (PF11_0150) shared specificity with rhomboid proteases from other organisms and was the only PfROM able to cleave apical membrane antigen 1 (AMA1). PfROM 1 and/or 4 was thus able to cleave diverse adhesins including TRAP, CTRP, MTRAP, PFF0800c, EBA-175, BAEBL, JESEBL, MAEBL, AMA1, Rh1, Rh2a, Rh2b, and Rh4, but not PTRAMP, and cleavage relied on the adhesin transmembrane domains. Swapping transmembrane regions between BAEBL and AMA1 switched the relative preferences of PfROMs 1 and 4 for these two substrates. Our analysis indicates that PfROMs 1 and 4 function with different substrate specificities that together constitute the specificity of TgROM5 to cleave diverse adhesins. This is the first enzymatic analysis of Plasmodium rhomboid proteases and suggests an involvement of PfROMs in all invasive stages of the malaria lifecycle, in both the vertebrate host and the mosquito vector.     

Outcomes among children and adults at risk of severe dengue in Sri Lanka: Opportunity for outpatient case management in countries with high disease burden

BackgroundHealthcare systems in dengue-endemic countries are often overburdened due to the high number of patients hospitalized according to dengue management guidelines. We systematically evaluated clinical outcomes in a large cohort of patients hospitalized with acute dengue to support triaging of patients to ambulatory versus inpatient management in the future.Methods/Principal findingsFrom June 2017- December 2018, we conducted surveillance among children and adults with fever within the prior 7 days who were hospitalized at the largest tertiary-care (1,800 bed) hospital in the Southern Province, Sri Lanka. Patients who developed platelet count ≤100,000/μL (threshold for hospital admission in Sri Lanka) and who met at least two clinical criteria consistent with dengue were eligible for enrollment. We confirmed acute dengue by testing sera collected at enrollment for dengue NS1 antigen or IgM antibodies. We defined primary outcomes as per the 1997 and 2009 World Health Organization (WHO) classification criteria: dengue hemorrhagic fever (DHF; WHO 1997), dengue shock syndrome (DSS; WHO 1997), and severe dengue (WHO 2009). Overall, 1064 patients were confirmed as having acute dengue: 318 (17.4%) by NS1 rapid antigen testing and 746 (40.7%) by IgM antibody testing. Of these 1064 patients, 994 (93.4%) were adults ≥18 years and 704 (66.2%) were male. The majority (56, 80%) of children and more than half of adults (544, 54.7%) developed DHF during hospitalization, while 6 (8.6%) children and 22 (2.2%) adults developed DSS. Overall, 10 (14.3%) children and 113 (11.4%) adults developed severe dengue. A total of 2 (0.2%) patients died during hospitalization.ConclusionsOne-half of patients hospitalized with acute dengue progressed to develop DHF and a very small number developed DSS or severe dengue. Developing an algorithm for triaging patients to ambulatory versus inpatient management should be the future goal to optimize utilization of healthcare resources in dengue-endemic countries.     

The barley Frost resistance-H2 locus

Frost resistance-H2 (Fr-H2) is a major QTL affecting freezing tolerance in barley, yet its molecular basis is still not clearly understood. To gain a better insight into the structural characterization of the locus, a high-resolution linkage map developed from the Nure × Tremois cross was initially implemented to map 13 loci which divided the 0.602 cM total genetic distance into ten recombination segments. A PCR-based screening was then applied to identify positive bacterial artificial chromosome (BAC) clones from two genomic libraries of the reference genotype Morex. Twenty-six overlapping BACs from the integrated physical-genetic map were 454 sequenced. Reads assembled in contigs were subsequently ordered, aligned and manually curated in 42 scaffolds. In a total of 1.47 Mbp, 58 protein-coding sequences were identified, 33 of which classified according to similarity with sequences in public databases. As three complete barley C-repeat Binding Factors (HvCBF) genes were newly identified, the locus contained13 full-length HvCBFs, four Related to AP2 Triticeae (RAPT) genes, and at least five CBF pseudogenes. The final overall assembly of Fr-H2 includes more than 90 % of target region: all genes were identified along the locus, and a general survey of Repetitive Elements obtained. We believe that this gold-standard sequence for the Morex Fr-H2 will be a useful genomic tool for structural and evolutionary comparisons with Fr-H2 in winter-hardy cultivars along with Fr-2 of other Triticeae crops.     

Backbone <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of the OB domain of the single stranded DNA-binding protein hSSB1 (NABP2/OBFC2B) and chemical shift mapping of the DNA-binding interface

Single-stranded DNA-binding proteins (SSBs) are highly important in DNA metabolism and play an essential role in all major DNA repair pathways. SSBs are generally characterised by the presence of an oligonucleotide binding (OB) fold which is able to recognise single-stranded DNA (ssDNA) with high affinity. We discovered two news SSBs in humans (hSSB1 and hSSB2) that both contain a single OB domain followed by a divergent spacer region and a charged C-terminus. We have extensively characterised one of these, hSSB1 (NABP2/OBFC2B), in numerous important DNA processing events such as, in DNA double-stranded break repair and in the response to oxidative DNA damage. Although the structure of hSSB1 bound to ssDNA has recently been determined using X-ray crystallography, the detailed atomic level mechanism of the interaction of hSSB1 with ssDNA in solution has not been established. In this study we report the solution-state backbone chemical shift assignments of the OB domain of hSSB1. In addition, we have utilized NMR to map the DNA-binding interface of hSSB1, revealing major differences between recognition of ssDNA under physiological conditions and in the recently determined crystal structure. Our NMR data in combination with further biophysical and biochemical experiments will allow us to address these discrepancies and shed light onto the structural basis of DNA-binding by hSSB1 in solution.     

Distribution,functional impact,and origin mechanisms of copy number variation in the barley genome

Background

There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys.

Results

A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley.

Conclusions

We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.     

Genomics-based high-resolution mapping of the BaMMV/BaYMV resistance gene rym11 in barley (Hordeum vulgare L.)

Soil-borne barley yellow mosaic virus disease, caused by different strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), is one of the most important diseases of winter barley (Hordeum vulgare L.) in Europe and East Asia. The recessive resistance gene rym11 located in the centromeric region of chromosome 4HL is effective against all so far known strains of BaMMV and BaYMV in Germany. In order to isolate this gene, a high-resolution mapping population (10,204 meiotic events) has been constructed. F₂ plants were screened with co-dominant flanking markers and segmental recombinant inbred lines (RILs) were tested for resistance to BaMMV under growth chamber and field conditions. Tightly linked markers were developed by exploiting (1) publicly available barley EST sequences, (2) employing barley synteny to rice, Brachypodium distachyon and sorghum and (3) using next-generation sequencing data of barley. Using this approach, the genetic interval was efficiently narrowed down from the initial 10.72 % recombination to 0.074 % recombination. A marker co-segregating with rym11 was developed providing the basis for gene isolation and efficient marker-assisted selection.     

The structural details of the interaction of single-stranded DNA binding protein hSSB2 (NABP1/OBFC2A) with UV-damaged DNA

Single-stranded DNA-binding proteins (SSBs) are required for all known DNA metabolic events such as DNA replication, recombination and repair. While a wealth of structural and functional data is available on the essential human SSB, hSSB1 (NABP2/OBFC2B), the close homolog hSSB2 (NABP1/OBFC2A) remains relatively uncharacterized. Both SSBs possess a well-structured OB (oligonucleotide/oligosaccharide-binding) domain that is able to recognize single-stranded DNA (ssDNA) followed by a flexible carboxyl-tail implicated in the interaction with other proteins. Despite the high sequence similarity of the OB domain, several recent studies have revealed distinct functional differences between hSSB1 and hSSB2. In this study, we show that hSSB2 is able to recognize cyclobutane pyrimidine dimers (CPD) that form in cellular DNA as a consequence of UV damage. Using a combination of biolayer interferometry and NMR, we determine the molecular details of the binding of the OB domain of hSSB2 to CPD-containing ssDNA, confirming the role of four key aromatic residues in hSSB2 (W59, Y78, W82, and Y89) that are also conserved in hSSB1. Our structural data thus demonstrate that ssDNA recognition by the OB fold of hSSB2 is highly similar to hSSB1, indicating that one SSB may be able to replace the other in any initial ssDNA binding event. However, any subsequent recruitment of other repair proteins most likely depends on the divergent carboxyl-tail and as such is likely to be different between hSSB1 and hSSB2.     

High-resolution mapping of the barley Ryd3 locus controlling tolerance to BYDV

Barley yellow dwarf disease (BYD) is transmitted by aphids and is caused by different strains of Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV). Economically it is one of the most important diseases of cereals worldwide. Besides chemical control of the vector, growing of tolerant/resistant cultivars is an effective way of protecting crops against BYD. The Ryd3 gene in barley (Hordeum vulgare L.) confers tolerance to BYDV-PAV and BYDV-MAV and the locus was previously mapped on the short arm of barley chromosome 6H near the centromere. We applied a strategy for high-resolution mapping and marker saturation at the Ryd3 locus by exploiting recent genomic tools available in barley. In a population of 3,210 F2 plants, 14 tightly linked markers were identified, including 10 that co-segregated with Ryd3. The centromeric region where Ryd3 is located suffers suppressed recombination or reduced recombination rate, suggesting potential problems in achieving (1) map-based cloning of Ryd3 and (2) marker selection of the resistance in breeding programmes without the introduction of undesirable traits via linkage drag.     

Backbone and side-chain 1H, 13C and 15N resonance assignments of the OB domain of the single stranded DNA binding protein from Sulfolobus solfataricus and chemical shift mapping of the DNA-binding interface

Single stranded DNA binding proteins (SSBs) are present in all known cellular organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has an unusual domain structure with a single DNA-binding oligonucleotide binding (OB) fold coupled to a flexible C-terminal tail. This ‘simple’ domain organisation differs significantly from other known SSBs, such as human replication protein A (RPA). However, it is conserved in another important human SSB, hSSB1, which we have recently discovered and shown to be essential in the DNA damage response. In this study we report the solution-state backbone and side-chain chemical shift assignments of the OB domain of SsoSSB. In addition, using the recently determined crystal structure, we have utilized NMR to reveal the DNA-binding interface of SsoSSB. These data will allow us to elucidate the structural basis of DNA-binding and shed light onto the molecular mechanism by which these ‘simple’ SSBs interact with single-stranded DNA.