Toxoplasma gondii, the causative agent of toxoplasmosis, is a widespread intracellular parasite able to infect virtually any nucleated cell. T. gondii infection of activated macrophages inhibits nitric oxide (NO) production; however, parasite effectors responsible for this block have not been defined. Macrophage populations are extremely heterogeneous, responding differently to stimuli and to parasite infection. Here we evaluated the inhibition of NO production caused by T. gondii infection of J774-A1 and RAW 264.7 macrophages and assessed the role of several known parasite virulence factors in this phenotype. Infection of activated macrophages from both macrophage lines reduced NO production, however, the mechanism of this decrease was different. Consistent with previous reports, infected J774-A1 macrophages had reduced iNOS expression and lower number of iNOS positive cells. In contrast, T. gondii infection of RAW 264.7 macrophages did not alter iNOS expression or the number of iNOS positive cells, and yet it led to lower levels of NO production. Deletion of a number of previously defined virulence factors including ROP kinases that disrupt innate immune factors, TgIST which blocks STAT1 activation, as well as the secretory trafficking proteins ASP5 and MYR1, did not alter the phenotype of decreased NO production. Taken together our findings indicate that T. gondii infection inhibits NO production of activated macrophages by different mechanisms that involve reduction of iNOS expression vs. iNOS impairment, and suggest that a novel parasite effector is involved in modulating this important host defense pathway.

The inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) protects a variety of cell types against neuronal apoptosis by binding to apolipoprotein E receptor 2 (ApoER2). The present study aimed to determine the association between PCSK9/ApoER2 signaling and neuronal apoptosis following middle cerebral artery occlusion (MCAO) injury in hyperlipidemic mice. For this purpose, C57BL/6 mice fed with a high-fat diet (HFD) for 6 weeks were exposed to NICAO. Subsequently, PCSK9 was inhibited by a lentiviral vector harboring short-hairpin RNA (shRNA) targeting PCSK9, which was stereotaxically injected into the cerebral cortex of mice. At 48 h post-ischemia, hematoxylin-eosin staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay were performed to determine cerebral tissue injury and apoptosis. PCSK9 and ApoER2 expression levels were assessed by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. The results indicated that hyperlipidemia and increased PCSK9 expression were evident in RFD mice. Cerebral histological injury and neuronal apoptosis, as well as PCSK9 and ApoER2 levels, which were increased upon ischemia in hyperlipidemic mice, were attenuated by PCSK9 shRNA treatment. These protective effects of PCSK9 shRNA interference were associated with decreased neuronal apoptosis and a reduced level of ApoER2 expression in the hippocampus and cortex. The data of the present study demonstrated that the PCSK9 shRNA-mediated anti-apoptotic effect induced by MCAO in hyperlipidemic mice is associated with ApoER2 downregulation, which may be a potential new therapy for stroke treatment in patients with hyperlipidemia.

Infection with the protozoan parasite, Toxoplasma gondii (T. gondii), has been associated with the increased risk for several psychiatric disorders. The exact mechanisms of a hypothesized contribution of T. gondii infection are poorly understood. The T. gondii genome contains two aromatic amino acid hydroxylase genes (AAH1 and AAH2) that encode proteins that can produce L-DOPA. One popular hypothesis posits that these encoded enzymes might influence dopamine (DA) production and hence DA synaptic transmission, leading to neurobehavioral abnormalities in the infected host. Prior studies have shown that deletion of these genes does not alter DA levels in the brain or exploratory activity in infected mice. However, possible effects of AAH gene deficiency on infection-induced brain and behavior alterations that are directly linked to DA synaptic transmission have not been evaluated. We found that chronic T. gondii infection of BALB/c mice leads to blunted response to amphetamine or cocaine and decreased expression of Dopamine Transporter (DAT) and Vesicular Monoamine Transporter 2 (VMAT2). Deletion of AAH2 had no effects on these changes in infected mice. Both wild type and Δaah2 strains produced comparable levels of neuroinflammation. Our findings demonstrate that AAH2 is not required for T. gondii infection-produced DA-dependent neurobehavioral abnormalities.

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host.

In their letter, McConkey et al. (1) suggest that differences in study design may have led to different results in our study compared with those of previous reports. In an effort to better understand these differences, we welcome the opportunity to provide more insight into the rationale for our study design and the reasoning behind the interpretations we reached. We stress at the outset that we had hoped to validate the previous findings that infection by Toxoplasma gondii leads to elevated dopamine production by dopaminergic cells in vitro and in chronically infected mice. In anticipation of such findings, we set out to disrupt the AAH2 gene, which had been implicated in this process by indirect data published previously by the McConkey group (2). Unfortunately, our studies failed to reproduce the original findings that infection increases dopamine production by using various parasite strains in different in vitro and in vivo models (3). In addition, review of the prior literature pertaining to the effects of T. gondii infection on elevated dopamine levels in vivo indicates that this pattern is highly variable (see points below). Because of the study design, the difficulties encountered in reproducing the original findings were not apparent until we had already generated the knockout and complement strains and begun testing them in parallel. This approach was chosen intentionally as an unbiased method for testing the hypothesis without a priori assumptions. We do not agree with the statement that it is essential to prove that our parental strain induces dopamine prior to engaging in gene knockout studies. Rather, our findings call into question whether the original findings are robust or generally reproducible. Although the letter by McConkey et al. suggests that our failure to observed elevated dopamine levels might be due to technical differences, we do not find this a compelling argument, as detailed in the following point-by-point response. (i) The authors claim that in their experiments, PC12 cells were kept competent to produce dopamine via attention to low passage number. We agree with the importance of this experimental detail. Our PC12 cells were expanded and frozen as low-passagenumber stocks shortly after being received from ATCC. In all experiments, cells were maintained for only 10 passages before being renewed from stock vials. (ii) The authors claim that our bradyzoite treatment, where we used high-pH growth medium to incubate infected PC12 cells, could quench dopamine production in these PC12 cells. Indeed, that is what we observed, a 25-fold decrease in total dopamine content produced by PC12 cells cultured under alkaline conditions. However, the result that was of interest to us was whether bradyzoites contribute to dopamine production in infected PC12 cells, as proposed by Prandovzsky et al. (2). If this prediction is correct, it stands to reason that the result should still hold even if dopamine production is globally lower. The fact that we still did not see a difference in overall dopamine content despite demonstrable evidence of differentiated bradyzoite-containing vacuoles inside PC12 cells challenges this hypothesis (3). (iii) The authors claim that their technique, soaking liberated tachyzoites in high-pH medium for 16 to 18 h and then infecting PC12 cells, differentiates the parasites into the bradyzoite stage without affecting the PC12 cells. We attempted to infect PC12 cells with bradyzoites that were liberated from high-pH-treated infected human foreskin fibroblast cells after 48 h, a time point when they stain positively for the Dolichos biflorus lectin (DBL), under conditions consistent with those of Prandovzsky et al. (2). However, at 48 h postinfection, the proportion of lectin-positive vacuoles was extremely low ( 12%). We expect that this resulted from parasites differentiating back into tachyzoites without continued pH stress to enforce the bradyzoite state. When we tried using high-pH treatment of extracellular parasites as reported by Prandovzsky et al. (2), we observed very poor viability, such that it was not possible to reliably infect PC12 cells. Hence, we modified the protocol to subject infected PC12 cells to high-pH medium, thereby inducing 100% DBL-positive vacuoles (i.e., bradyzoites). Under these conditions, we failed to see dopamine changes (3). (iv) The authors claim that in the absence of viability data, it cannot be excluded that the 25-fold dopamine decrease in alkaline PC12 cells could be due to cell death. PC12 cells were counted by light microscopy before being harvested into perchloric acid buffer for high-performance liquid chromatography, and the level of dopamine per 10 cells was determined on the basis of visual identification of intact cells. If alkaline treatment led to increased mortality of PC12 cells, this would be corrected for by this visual analysis. (v) The authors rightly point out that parasite strain differences might exist in the effects on dopamine and behavior and also point out that our strain, Pru ku80 hxg, has not been previously demonstrated to induce elevated dopamine prior to gene knockout. However, Pru ku80 hxg was not the only strain used in our study (3). We observed the same absence of dopamine change in vitro when using ME49 (unpublished data). In vivo, we demonstrated no change in brain dopamine not only in mice infected with Pru ku80 hxg but also in mice infected with ME49 and the original C56 strain used by Stibbs (4). Thus, the failure to observe

ABSTRACT Toxoplasma gondii infection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo . Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo , even when AAH2 was overexpressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii , since AAH1 is essential for growth in nondopaminergic cells.

ABSTRACT Light microscopy and deep-etch electron microscopy were used to visualize triacylglyceride (TAG)-filled lipid bodies (LBs) of the green eukaryotic soil alga Chlamydomonas reinhardtii , a model organism for biodiesel production. Cells growing in nitrogen-replete media contain small cytoplasmic lipid bodies (α-cyto-LBs) and small chloroplast plastoglobules. When starved for N, β-cyto-LB formation is massively stimulated. β-Cyto-LBs are intimately associated with both the endoplasmic reticulum membrane and the outer membrane of the chloroplast envelope, suggesting a model for the active participation of both organelles in β-cyto-LB biosynthesis and packaging. When sta6 mutant cells, blocked in starch biosynthesis, are N starved, they produce β-cyto-LBs and also chloroplast LBs (cpst-LBs) that are at least 10 times larger than plastoglobules and eventually engorge the chloroplast stroma. Production of β-cyto-LBs and cpst-LBs under the conditions we used is dependent on exogenous 20 mM acetate. We propose that the greater TAG yields reported for N-starved sta6 cells can be attributed to the strain's ability to produce cpst-LBs, a capacity that is lost when the mutant is complemented by a STA6 transgene. Provision of a 20 mM acetate “boost” during N starvation generates sta6 cells that become so engorged with LBs—at the expense of cytoplasm and most organelles—that they float on water even when centrifuged. This property could be a desirable feature for algal harvesting during biodiesel production.

ABSTRACTWhen the unicellular green soil algaChlamydomonas reinhardtiiis deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in thesta6mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C16and C18) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C18:1). The FFA are ∼50% C16and ∼50% C18. The LB-derived TAG yield from a liter ofsta6cells at 107cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production inC. reinhardtiihas the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.

Acaryochloris marina is a unique cyanobacterium that is able to produce chlorophyll d as its primary photosynthetic pigment and thus efficiently use far-red light for photosynthesis. Acaryochloris species have been isolated from marine environments in association with other oxygenic phototrophs, which may have driven the niche-filling introduction of chlorophyll d. To investigate these unique adaptations, we have sequenced the complete genome of A. marina. The DNA content of A. marina is composed of 8.3 million base pairs, which is among the largest bacterial genomes sequenced thus far. This large array of genomic data is distributed into nine single-copy plasmids that code for >25% of the putative ORFs. Heavy duplication of genes related to DNA repair and recombination (primarily recA) and transposable elements could account for genetic mobility and genome expansion. We discuss points of interest for the biosynthesis of the unusual pigments chlorophyll d and alpha-carotene and genes responsible for previously studied phycobilin aggregates. Our analysis also reveals that A. marina carries a unique complement of genes for these phycobiliproteins in relation to those coding for antenna proteins related to those in Prochlorococcus species. The global replacement of major photosynthetic pigments appears to have incurred only minimal specializations in reaction center proteins to accommodate these alternate pigments. These features clearly show that the genus Acaryochloris is a fitting candidate for understanding genome expansion, gene acquisition, ecological adaptation, and photosystem modification in the cyanobacteria.

ABSTRACT Despite the fact that heliobacteria are the only phototrophic representatives of the bacterial phylum Firmicutes , genomic analyses of these organisms have yet to be reported. Here we describe the complete sequence and analysis of the genome of Heliobacterium modesticaldum , a thermophilic species belonging to this unique group of phototrophs. The genome is a single 3.1-Mb circular chromosome containing 3,138 open reading frames. As suspected from physiological studies of heliobacteria that have failed to show photoautotrophic growth, genes encoding enzymes for known autotrophic pathways in other phototrophic organisms, including ribulose bisphosphate carboxylase (Calvin cycle), citrate lyase (reverse citric acid cycle), and malyl coenzyme A lyase (3-hydroxypropionate pathway), are not present in the H. modesticaldum genome. Thus, heliobacteria appear to be the only known anaerobic anoxygenic phototrophs that are not capable of autotrophy. Although for some cellular activities, such as nitrogen fixation, there is a full complement of genes in H. modesticaldum , other processes, including carbon metabolism and endosporulation, are more genetically streamlined than they are in most other low-G+C gram-positive bacteria. Moreover, several genes encoding photosynthetic functions in phototrophic purple bacteria are not present in the heliobacteria. In contrast to the nutritional flexibility of many anoxygenic phototrophs, the complete genome sequence of H. modesticaldum reveals an organism with a notable degree of metabolic specialization and genomic reduction.