Erick R. James

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing.

Current sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the worlds oceans.

ABCA1 mRNA and Protein Distribution Patterns Predict Multiple Different Roles and Levels of Regulation

Mutations in ABCA1 cause the allelic disorders familial hypolipoproteinemia and Tangier Disease. To identify where ABCA1 was likely to have a functional role, we determined the cellular and tissue-specific patterns of murine ABCA1 expression. RT-PCR and Western blot analysis on dissected murine tissues demonstrated broad expression of ABCA1 mRNA and protein in many tissues with prominent protein expression in liver, testis, and adrenal tissue. In situ hybridization and immunohistochemistry experiments demonstrated specific patterns of ABCA1 expression at the cellular level, with hepatocytes, the epithelial lining of the digestive system and bladder, the proximal convoluted tubule of the kidney, and Purkinje and cortical pyramidal neurons containing abundant ABCA1 protein. Significant discordance between relative mRNA and protein expression patterns suggests the possibility of post-transcriptional regulation of ABCA1 expression in selected cells or tissues. We also show that ABCA1 protein levels are up-regulated specifically in the liver after exposure to an atherogenic diet for 7 days, supporting a major role for the liver in dietary modulation of HDL-C levels. Our observations show that ABCA1 is expressed in a pattern consistent with its role in HDL-C metabolism. Additionally, ABCA1 may have important functional roles in other cell types independent of HDL-C regulation.

Increased ABCA1 activity protects against atherosclerosis.

The ABC transporter ABCA1 plays a key role in the first steps of the reverse cholesterol transport pathway by mediating lipid efflux from macrophages. Previously, it was demonstrated that human ABCA1 overexpression in vivo in transgenic mice results in a mild elevation of plasma HDL levels and increased efflux of cholesterol from macrophages. In this study, we determined the effect of overexpression of ABCA1 on atherosclerosis development. Human ABCA1 transgenic mice (BAC(+)) were crossed with ApoE(-/-) mice, a strain that spontaneously develop atherosclerotic lesions. BAC(+)ApoE(-/-) mice developed dramatically smaller, less-complex lesions as compared with their ApoE(-/-) counterparts. In addition, there was increased efflux of cholesterol from macrophages isolated from the BAC(+)ApoE(-/-) mice. Although the increase in plasma HDL cholesterol levels was small, HDL particles from BAC(+)ApoE(-/-) mice were significantly better acceptors of cholesterol. Lipid analysis of HDL particles from BAC(+)ApoE(-/-) mice revealed an increase in phospholipid levels, which was correlated significantly with their ability to enhance cholesterol efflux.

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: New linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Macrophage ATP-binding cassette transporter A1 overexpression inhibits atherosclerotic lesion progression in low-density lipoprotein receptor knockout mice.

Background—ATP-binding cassette transporter A1 (ABCA1) is a key regulator of cellular cholesterol and phospholipid transport. Previously, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis in low-density lipoprotein receptor knockout (LDLr−/−) mice. However, the possibly beneficial effects of specific upregulation of macrophage ABCA1 on atherogenesis are still unknown. Methods and Results—Chimeras that specifically overexpress ABCA1 in macrophages were generated by transplantation of bone marrow from human ABCA1 bacterial artificial chromosome (BAC) transgenic mice into LDLr−/− mice. Peritoneal macrophages isolated from the ABCA1 BAC → LDLr−/− chimeras exhibited a 60% (P=0.0006) increase in cholesterol efflux to apolipoprotein AI. To induce atherosclerosis, the mice were fed a Western-type diet containing 0.25% cholesterol and 15% fat for 9, 12, and 15 weeks, allowing analysis of effects on initial lesion development as well as advanced lesions. No significant effect of macrophage ABCA1 overexpression was observed on atherosclerotic lesion size after 9 weeks on the Western-type diet (245±36×103 &mgr;m2 in ABCA1 BAC → LDLr−/− mice versus 210±20×103 &mgr;m2 in controls). However, after 12 weeks, the mean atherosclerotic lesion area in ABCA1 BAC → LDLr−/− mice remained only 164±15×103 &mgr;m2 (P=0.0008) compared with 513±56×103 &mgr;m2 in controls (3.1-fold lower). Also, after 15 weeks on the diet, lesions in mice transplanted with ABCA1 overexpressing bone marrow were still 1.6-fold smaller (393±27×103 &mgr;m2 compared with 640±59×103 &mgr;m2 in control transplanted mice; P=0.0015). Conclusion—ABCA1 upregulation in macrophages inhibits the progression of atherosclerotic lesions.

Protein kinase A site-specific phosphorylation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux

ATP-binding cassette A1 (ABCA1) is a key mediator of cholesterol and phospholipid efflux to apolipoprotein particles. We show that ABCA1 is a constitutively phosphorylated protein in both RAW macrophages and in a human embryonic kidney cell line expressing ABCA1. Furthermore, we demonstrate that phosphorylation of ABCA1 is mediated by protein kinase A (PKA) or a PKA-like kinasein vivo. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assays, we show that Ser-1042 and Ser-2054, located in the nucleotide binding domains of ABCA1, are major phosphorylation sites for PKA. ApoA-I-dependent phospholipid efflux was decreased significantly by mutation of Ser-2054 alone and Ser-1042/Ser-2054 but was not significantly impaired with Ser-1042 alone. The mechanism by which ABCA1 phosphorylation affected ApoA-I-dependent phospholipid efflux did not involve either alterations in ApoA-I binding or changes in ABCA1 protein stability. These studies demonstrate a novel serine (Ser-2054) on the ABCA1 protein crucial for PKA phosphorylation and for regulation of ABCA1 transporter activity.

ABCA1 Regulatory Variants Influence Coronary Artery Disease Independent of Effects on Plasma Lipid Levels

The authors have previously shown that individuals heterozygous for ABCA1 mutations have decreased high density lipoprotein cholesterol, increased triglycerides and an increased frequency of coronary artery disease (CAD), and that single nucleotide polymorphisms (SNPs) in the coding region of the ABCA1 gene significantly impact plasma lipid levels and the severity of CAD in the general population. They have now identified several SNPs in non‐coding regions of ABCA1 which may be important for the appropriate regulation of ABCA1 expression (i.e. in the promoter, intron 1 and the 5′ untranslated region), and have examined the phenotypic effects of these SNPs in the REGRESS population. Out of 12 SNPs, four were associated with a clinical outcome. A threefold increase in coronary events with an increased family history of CAD was evident for the G‐191C variant. Similarly, the C69T SNP was associated with a twofold increase in events. In contrast, the C‐17G was associated with a decrease in coronary events and the InsG319 was associated with less atherosclerosis. For all these SNPs, the changes in atherosclerosis and CAD occurred without detectable changes in plasma lipid levels. These data suggest that common variation in non‐coding regions of ABCA1 may significantly alter the severity of atherosclerosis, without necessarily influencing plasma lipid levels.

Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments

Background Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments. Methodology/Principal Findings In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species. Conclusions/Significance COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.

Identification of an Atypical Strain of Toxoplasma gondii as the Cause of a Waterborne Outbreak of Toxoplasmosis in Santa Isabel do Ivai, Brazil

Multilocus DNA sequencing has identified a nonarchetypal strain of Toxoplasma gondii as the causal agent of a waterborne outbreak in Brazil in 2001. The strain, isolated from a water supply epidemiologically linked to the outbreak, was virulent to mice, and it has previously been identified as BrI. Using a serologic assay that detects strain-specific antibodies, we found that 13 (65%) of 20 individuals who were immunoglobulin (Ig) M positive during the outbreak possessed the same serotype as mice infected with the purported epidemic strain. The remaining 7 individuals, plus additional IgM-negative, IgG-positive individuals, possessed 1 of 4 novel serotypes, the most common of which matched the serotype of mice infected with strains isolated from chickens foraging near the outbreak site. The latter strains likely reflect the genetic diversity of T. gondii circulating in highly endemic regions of Brazil. The serotyping assay proved a useful tool for identification of specific individuals infected with the outbreak agent.

A bacterial proteorhodopsin proton pump in marine eukaryotes

Proteorhodopsins are light-driven proton pumps involved in widespread phototrophy. Discovered in marine proteobacteria just 10 years ago, proteorhodopsins are now known to have been spread by lateral gene transfer across diverse prokaryotes, but are curiously absent from eukaryotes. In this study, we show that proteorhodopsins have been acquired by horizontal gene transfer from bacteria at least twice independently in dinoflagellate protists. We find that in the marine predator Oxyrrhis marina, proteorhodopsin is indeed the most abundantly expressed nuclear gene and its product localizes to discrete cytoplasmic structures suggestive of the endomembrane system. To date, photosystems I and II have been the only known mechanism for transducing solar energy in eukaryotes; however, it now appears that some abundant zooplankton use this alternative pathway to harness light to power biological functions.