Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 Å resolution

The R. prowazekii 3-ketoacyl-(acyl-carrier-protein) reductase is similar to those from other prokaryotic pathogens but differs significantly from the mammalian orthologue, strengthening its case as a potential drug target

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatio-temporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium. IMPORTANCE Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.Peer reviewe

Multiple Roles for the Non-Coding RNA SRA in Regulation of Adipogenesis and Insulin Sensitivity

Peroxisome proliferator-activated receptor-γ (PPARγ) is a master transcriptional regulator of adipogenesis. Hence, the identification of PPARγ coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA, Steroid receptor RNA Activator (SRA), associates with PPARγ and coactivates PPARγ-dependent reporter gene expression. Overexpression of SRA in ST2 mesenchymal precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes involved in the cell cycle, and insulin and TNFα signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPARγ. SRA in adipocytes increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the expression of adipocyte-related inflammatory genes and TNFα-induced phosphorylation of c-Jun NH2-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Endogenous tagging and generation of knockouts in <i>T</i>. <i>gondii</i>.

<p><b>A</b>. Schematic of the CRISPR/Cas9 tagging system. Tagging plasmids were generated with various tags (green box) flanked by common ends (red and black boxes) and including a common stop codon (gray box) followed by the <i>HXGPRT</i> 3’ UTR (yellow box) and the selectable marker HXGPRT. Amplification of this central region with primers that contained short homology regions HR1 (purple box) and HR2 (blue box) together with the common flanks (red and black boxes) generated products for gene-specific tagging. Co-transfection of these amplicons with a CRISPR/Cas9 plasmid bearing the gene-specific single guide RNA (sgRNA3’) was used to add an epitope tag (green box) at the C-terminus of the endogenous locus. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#ppat.1006379.s006" target="_blank">S1 Fig</a> for more details. <b>B</b>. Localization of CaM1, CaM2 and CaM3 containing C-terminal 6HA tags. Detected with mouse anti-HA (green) and rabbit anti-GAP45 (red). Scale bar, 2 μM. <b>C</b>. Schematic of the double CRISPR/Cas9 gRNA system used for generation of clean knockouts using two sgRNAs matching the 5’ and 3’ ends of the coding sequence. The entire coding sequence was replaced by the DHFR marker flanked by short homology regions (HR3, red; HR2, blue). Primers (p) used for diagnostic PCR. <b>D</b>. Diagnostic PCR of knockouts compared to the parental ku80^KO line. <i>CDPK1</i>, PCR control. <b>E.</b> Plaque numbers formed by the knockouts compared to the parental ku80^KO line. ns, not significant, analyzed by one-way ANOVA.</p

Analysis of egress, invasion, and motility in parental and mutant lines.

<p><b>A</b>. Parasites grown for 30 hr ± IAA (500 μM vs 0.1% ethanol) were stimulated with 3 μM A23187 to simulate egress. Rabbit anti-GRA7 (red) and mouse anti-IMC1 (green) antibodies were used to distinguish intact vs. egressed vacuoles. *** <i>P ≤ 0</i>.<i>0001</i>, significant for the time points of 2, 5, 10 and 15 min, but not significant for 0 and 20 min. Scale bar, 5 μM. <b>B</b>. Quantitative analysis of invasion by parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) and used to challenge fresh HFF monolayers on coverslips for 20 min. Extracellular parasites (invaded) were distinguished from those that remained extracellular (attached) by differential IFA staining (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#sec002" target="_blank">methods</a>). *** <i>P</i> ≤ 0.0001. <b>C.</b> Evaluation of cell entry past the moving junction. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) were used to challenge fresh HFF monolayers on coverslips for 3 min, fixed and stained with rabbit anti-RON4 (green) and mouse anti-SAG1 (red) without permeabilization. Parasites with RON4 dots were considered to be apically attached (red column), and parasites with RON4 positive rings were classified as partially invaded (green column). *** <i>P ≤ 0</i>.<i>0001</i>. Scale bar, 2 μM. <b>D</b>. Parasite motility as monitored by video microscopy. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) were allowed to glide on serum-coated coverslips. Time-lapse video microscopy was used to score different motile behaviors. *** <i>P</i> ≤ 0.0001, the cam2^KOCaM1-AID line showed significant decrease in twirling and increase non-productive movement when grown in +IAA <i>vs</i>. -IAA or the TIR1 parental line, **, <i>P</i> ≤ 0.0001, the CaM3-AID line showed a significant decrease in twirling and increase in circling when grown in +IAA <i>vs</i>. -IAA or the TIR1 parental line. Panels <b>A</b>, <b>B</b>, <b>C</b>, <b>D</b> represent means ± S.D. from three independent experiments with triplicates for each (n = 9). Two-way ANOVA with Tukey’s multiple comparison test for <b>A</b>, <b>C</b> and <b>D</b>, and one-way ANOVA with Tukey’s multiple comparison test for <b>B</b>.</p

Analysis of parasite replication, conoid protrusion, apical organelle distribution and secretion in parental and mutant lines.

<p><b>A</b>. Parasite replication after 24 hr incubation with ± IAA (500 μM vs 0.1% ethanol). ns, not significant. <b>B</b>. Proportion of parasites with extruded conoid. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol), stimulated with 3 μM A23187 or DMSO vehicle control for 10 min. ns, not significant. <b>C and D</b>. Distribution of MIC2 (mouse anti-MIC2 (green) and ROP5 (rabbit annti-ROP5 (green) upon depletion of AID fusion proteins. Parasites grown ± IAA (500 μM vs 0.1% ethanol) for 24 hr in HFF monolayers and stained for IFA. Parasites were counterstained with mouse anti-IMC1 (red) or rabbit anti-GAP45 antibodies (green). Scale bar, 2 μM. <b>E.</b> Quantification of micronemal secretion using MIC2-GLuc-myc reporter lines. Parasites were grown for 2 days ±IAA (500 μM vs 0.1% ethanol), stimulated with 1% ethanol—1% BSA and secretion was monitored by releases of luciferase (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#sec002" target="_blank">methods</a>). Relative Luminescence Unit (RLU)<b>.</b> ns, not significant. <b>F and G.</b> Detection of rhoptry secretion by ROP1 staining. Parasites were grown for 2 days ± IAA (500 μM vs 0.1% ethanol), harvested and used to detect formation of evacuoles (arrows) on fresh monolayers of HFF cells in the presence of cytochalasin. Parasites were counted from triplicate samples on three separate experiments and ratios of parasites associated with evacuoles in were plotted. Scale bar, 5 μm. Panels <b>A</b>, <b>B</b>, <b>E</b>, <b>F, G</b> mean ± S.D. from three independent experiments with triplicates for each (n = 9). One-way ANOVA with Tukey’s multiple comparison test for <b>B</b> and <b>E</b> and two-way ANOVA with Tukey’s multiple comparison test for pair-wise multiple comparisons across each vacuole size for <b>A</b>, Man-Whitney non-parametric test for <b>F</b> and <b>G</b>.</p

Phenotypic comparison among <i>T</i>. <i>gondii</i> mutant lines.

<p>Phenotypic comparison among <i>T</i>. <i>gondii</i> mutant lines.</p

Generation of AID tagged lines in the TIR parental line of <i>T</i>. <i>gondii</i>.

<p><b>A.</b> Western blot analysis using antibodies to detect CaM1-AID or CaM3-AID (mouse anti-HA to the AID-3HA tag), TIR1-3Flag (rat anti-Flag) and aldolase (rabbit anti-aldolase, ALD). <b>B and C.</b> Degradation of AID tagged proteins in cam2^KO<i>/</i>CaM1-AID (<b>B</b>) and CaM3-AID (<b>C</b>) lines after addition of auxin (500 μM IAA) for different time periods. Mock indicates parasites grown with 0.1% ethanol for 36 hr. CaM1-AID or CaM3-AID proteins were detected with mouse anti-HA and rabbit anti-aldolase (ALD) antibodies served as a loading control. Band intensities were analyzed by ImageJ, and ratios of anti-HA vs. anti-ALD signal were calculated (HA/ALD) and expressed as a percentage of the mock treatment (i.e. 100%). <b>D and E</b>. Degradation of AID tagged proteins in cam2^KO<i>/</i>CaM1-AID (D) and CaM3-AID (E) parasites after 24 hr incubation with 500 μM IAA (+IAA) or ethanol vehicle 0.1% (-IAA). CaM1-AID or CaM3-AID proteins were detected with mouse anti-HA (green) and rabbit GAP45 (red) antibodies served as a control to label the parasite. Scale bar, 2 μM. <b>F</b>. Plaque formation by parasites grown on HFF monolayers. Scale bar, 0.5 cm. Insert images in the CaM3-AID line, scale bar (red) = 1 mm. <b>G</b>. Measurement of plaque numbers and sizes for the CaM3-AID line treated with and without auxin. N≥ 25, ***, <i>P</i> < 0.0001. Mann Whitney non-parametric test.</p