Michael R. Chase

Epstein–Barr virus and virus human protein interaction maps

A comprehensive mapping of interactions among Epstein–Barr virus (EBV) proteins and interactions of EBV proteins with human proteins should provide specific hypotheses and a broad perspective on EBV strategies for replication and persistence. Interactions of EBV proteins with each other and with human proteins were assessed by using a stringent high-throughput yeast two-hybrid system. Overall, 43 interactions between EBV proteins and 173 interactions between EBV and human proteins were identified. EBV–EBV and EBV–human protein interaction, or “interactome” maps provided a framework for hypotheses of protein function. For example, LF2, an EBV protein of unknown function interacted with the EBV immediate early R transactivator (Rta) and was found to inhibit Rta transactivation. From a broader perspective, EBV genes can be divided into two evolutionary classes, “core” genes, which are conserved across all herpesviruses and subfamily specific, or “noncore” genes. Our EBV–EBV interactome map is enriched for interactions among proteins in the same evolutionary class. Furthermore, human proteins targeted by EBV proteins were enriched for highly connected or “hub” proteins and for proteins with relatively short paths to all other proteins in the human interactome network. Targeting of hubs might be an efficient mechanism for EBV reorganization of cellular processes.

Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection

Tuberculosis poses a global health emergency, which has been compounded by the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains. We used whole-genome sequencing to compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent or reactivated disease. We sequenced 33 Mtb isolates from nine macaques with an average genome coverage of 93% and an average read depth of 117×. Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states. We found a similar mutation rate during latency as during active disease or in a logarithmically growing culture over the same period of time. The pattern of polymorphisms suggests that the mutational burden in vivo is because of oxidative DNA damage. We show that Mtb continues to acquire mutations during disease latency, which may explain why isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of isoniazid resistance.

Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments

T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.

High abundance synovial fluid proteome: distinct profiles in health and osteoarthritis

The development of increasingly high-throughput and sensitive mass spectroscopy-based proteomic techniques provides new opportunities to examine the physiology and pathophysiology of many biologic fluids and tissues. The purpose of this study was to determine protein expression profiles of high-abundance synovial fluid (SF) proteins in health and in the prevalent joint disease osteoarthritis (OA). A cross-sectional study of 62 patients with early OA (n = 21), patients with late OA (n = 21), and control individuals (n = 20) was conducted. SF proteins were separated by using one-dimensional PAGE, and the in-gel digested proteins were analyzed by electrospray ionization tandem mass spectrometry. A total of 362 spots were examined and 135 high-abundance SF proteins were identified as being expressed across all three study cohorts. A total of 135 SF proteins were identified. Eighteen proteins were found to be significantly differentially expressed between control individuals and OA patients. Two subsets of OA that are not dependent on disease duration were identified using unsupervised analysis of the data. Several novel SF proteins were also identified. Our analyses demonstrate no disease duration-dependent differences in abundant protein composition of SF in OA, and we clearly identified two previously unappreciated yet distinct subsets of protein profiles in this disease cohort. Additionally, our findings reveal novel abundant protein species in healthy SF whose functional contribution to SF physiology was not previously recognized. Finally, our studies identify candidate biomarkers for OA with potential for use as highly sensitive and specific tests for diagnostic purposes or for evaluating therapeutic response.

Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata.

Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.

POPULATION DIFFERENTIATION DECREASES WITH DEPTH IN DEEP-SEA BIVALVES

Abstract The deep sea is the largest ecosystem on Earth. Recent exploration has revealed that it supports a highly diverse and endemic benthic invertebrate fauna, yet the evolutionary processes that generate this remarkable species richness are virtually unknown. Environmental heterogeneity, topographic complexity, and morphological divergence all tend to decrease with depth, suggesting that the potential for population differentiation may decrease with depth.To test this hypothesis, we use mitochondrial DNA (16S rRNA gene) to examine patterns of population differentiationin four species of protobranch bivalves (Nuculoma similis, Deminucula atacellana, Malletia abyssorum, and Ledellaultima) distributed along a depth gradient in the western North Atlantic. We sequenced 268 individuals from formalinfixed samples and found 45 haplotypes. The level of sequence divergence among haplotypes within species was similar, but shifted from between populations at bathyal depths to within populations at abyssal depths. Levels of population structure as measured by ST were considerably greater in the upper bathyal species (N. similis= 0.755 and D. atacellana= 0.931; 530–3834 m) than in the lower bathyal/abyssal species (M. abyssorum= 0.071 and L. ultima= 0.045; 2864–4970 m). Pairwise genetic distances among the samples within each species also decreased with depth. Population trees (UPGMA) based on modified coancestry coefficients and nested clade analysis both indicated strong population‐level divergence in the two upper bathyal species but little for the deeper species. The population genetic structure in these protobranch bivalves parallels depth‐related morphological divergence observed in deep‐sea gastropods.The higher level of genetic and morphological divergence, coupled with the strong biotic and abiotic heterogeneityal bathyal depths, suggests this region may be an active area of species formation. We suggest that the steep, topographically complex, and dynamic bathyal zone, which stretches as a narrow band along continental margins, plays a more important role in the evolutionary radiation of the deep‐sea fauna than the much more extensive abyss.

Population genetics of Cordia alliodora (Boraginaceae), a neotropical tree. 2. Mating system.

A multilocus mixed mating model was used to evaluate the mating system of a natural population of Cordia alliodora (Boraginaceae), a neotropical tree. The population was highly outcrossed (tin = 0.966 ? 0.027), in agreement with results from controlled crosses. Departures from the mixed mating model were evident, suggesting some nonrandom, correlated mating. Pollen pool heterogeneity and variation in estimates of individual outcrossing rates indicated that the population may be genetically substructured. Individual outcrossing rates obtained for the samples taken from within different parts of the same tree indicated reduced levels of outcrossing due to limited sampling of the pollen pool. The incompatibility mechanism in C. alliodora, combined with variation in flowering and stand density, appears to lead to both temporal and spatial substructuring of the population.

The genome and structural proteome of an ocean siphovirus: a new window into the cyanobacterial 'mobilome'

Prochlorococcus, an abundant phototroph in the oceans, are infected by members of three families of viruses: myo-, podo- and siphoviruses. Genomes of myo- and podoviruses isolated on Prochlorococcus contain DNA replication machinery and virion structural genes homologous to those from coliphages T4 and T7 respectively. They also contain a suite of genes of cyanobacterial origin, most notably photosynthesis genes, which are expressed during infection and appear integral to the evolutionary trajectory of both host and phage. Here we present the first genome of a cyanobacterial siphovirus, P-SS2, which was isolated from Atlantic slope waters using a Prochlorococcus host (MIT9313). The P-SS2 genome is larger than, and considerably divergent from, previously sequenced siphoviruses. It appears most closely related to lambdoid siphoviruses, with which it shares 13 functional homologues. The ∼108 kb P-SS2 genome encodes 131 predicted proteins and notably lacks photosynthesis genes which have consistently been found in other marine cyanophage, but does contain 14 other cyanobacterial homologues. While only six structural proteins were identified from the genome sequence, 35 proteins were detected experimentally; these mapped onto capsid and tail structural modules in the genome. P-SS2 is potentially capable of integration into its host as inferred from bioinformatically identified genetic machinery int, bet, exo and a 53 bp attachment site. The host attachment site appears to be a genomic island that is tied to insertion sequence (IS) activity that could facilitate mobility of a gene involved in the nitrogen-stress response. The homologous region and a secondary IS-element hot-spot in Synechococcus RS9917 are further evidence of IS-mediated genome evolution coincident with a probable relic prophage integration event. This siphovirus genome provides a glimpse into the biology of a deep-photic zone phage as well as the ocean cyanobacterial prophage and IS element ‘mobilome’.

Bathymetric and geographic population structure in the pan‐Atlantic deep‐sea bivalve Deminucula atacellana (Schenck, 1939)

The deep‐sea soft‐sediment environment hosts a diverse and highly endemic fauna of uncertain origin. We know little about how this fauna evolved because geographic patterns of genetic variation, the essential information for inferring patterns of population differentiation and speciation are poorly understood. Using formalin‐fixed specimens from archival collections, we quantify patterns of genetic variation in the protobranch bivalve Deminucula atacellana, a species widespread throughout the Atlantic Ocean at bathyal and abyssal depths. Samples were taken from 18 localities in the North American, West European and Argentine basins. A hypervariable region of mitochondrial 16S rDNA was amplified by polymerase chain reaction (PCR) and sequenced from 130 individuals revealing 21 haplotypes. Except for several important exceptions, haplotypes are unique to each basin. Overall gene diversity is high (h = 0.73) with pronounced population structure (ΦST = 0.877) and highly significant geographic associations (P < 0.0001). Sequences cluster into four major clades corresponding to differences in geography and depth. Genetic divergence was much greater among populations at different depths within the same basin, than among those at similar depths but separated by thousands of kilometres. Isolation by distance probably explains much of the interbasin variation. Depth‐related divergence may reflect historical patterns of colonization or strong environmental selective gradients. Broadly distributed deep‐sea organisms can possess highly genetically divergent populations, despite the lack of any morphological divergence.

EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity

Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall.