De novo transcriptome reconstruction and annotation of the Egyptian rousette bat

Background The Egyptian Rousette bat (Rousettus aegyptiacus), a common fruit bat species found throughout Africa and the Middle East, was recently identified as a natural reservoir host of Marburg virus. With Ebola virus, Marburg virus is a member of the family Filoviridae that causes severe hemorrhagic fever disease in humans and nonhuman primates, but results in little to no pathological consequences in bats. Understanding host-pathogen interactions within reservoir host species and how it differs from hosts that experience severe disease is an important aspect of evaluating viral pathogenesis and developing novel therapeutics and methods of prevention. Results Progress in studying bat reservoir host responses to virus infection is hampered by the lack of host-specific reagents required for immunological studies. In order to establish a basis for the design of reagents, we sequenced, assembled, and annotated the R. aegyptiacus transcriptome. We performed de novo transcriptome assembly using deep RNA sequencing data from 11 distinct tissues from one male and one female bat. We observed high similarity between this transcriptome and those available from other bat species. Gene expression analysis demonstrated clustering of expression profiles by tissue, where we also identified enrichment of tissue-specific gene ontology terms. In addition, we identified and experimentally validated the expression of novel coding transcripts that may be specific to this species. Conclusion We comprehensively characterized the R. aegyptiacus transcriptome de novo. This transcriptome will be an important resource for understanding bat immunology, physiology, disease pathogenesis, and virus transmission

Sox11 Reduces Caspase-6 Cleavage and Activity.

The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP) family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS) all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11

Sox11 reduces caspase-6 activity to control levels.

<p>(A) Lysates from HEK293FT cells were collected and assayed for caspase-6 activity via a luciferase based substrate assay system. NB caspase-6 Δpro refers to caspase-6 Δprodomain. The western blot shows caspase-6 activity is decreased in the presence of sox11. Caspase-6 activity was reduced to control levels by sox11. Statistical analysis showed this reduction was very significant (n = 3, one-way anova, **** p<0001). (B) Negative control experiments were performed using two other proteins, FEZ1 and DISC1. Neither FEZ1 nor DISC1 caused a significant decrease in caspase-6 activity but a one-way anova, followed by a Sidak’s multiple comparsion test showed DISC1 caused a slight increase in caspase-6 activity (n = 3, **** p<0001, *<0.05) (C) RNA was extracted from HEK293FT cells transfected for 24hours with indicated plasmids. Reverse transcription and PCR amplification using caspase-6 primers showed no significant change in mRNA levels between single and co-transfected samples (compare lanes 2, 3, and 4 with 6, 7, and 8, respectively). GAPDH levels were equal across all samples tested (n = 3). (D) A luciferase based reporter assay was used to assay sox11 transcriptional activity in the presence of caspase-6. Sox11 transactivation potential was not significantly affected by caspase-6 or caspase-6 C163A.</p

Sox11 is protective against apoptotic insult and other sox proteins can also reduce caspase-6 activity.

<p>(A) Mouse cortical primary neurons were transfected with either sox11 or sox11 ΔNLS. 24 hours post-transfection, neurons were either treated with toxic agents or subjected to serum starvation for a further 24 hours. Neurons were fixed and stained and assessed for cell death via nuclear condensation (n = 8, one-way anova, Sidak’s multiple comparison test ****p<0.0001, **p<0.01). (B) Caspase-6 activity was measured using a luciferase-based substrate assay from cells transfected with indicated plasmids. Sox11 reduced caspase-6 activity to control levels. Sox4 and sox7 partially reduced caspase-6 activation (n = 2, one-way anova, Sidak’s multiple comparison test ****p<0.0001, **p<0.01).</p

Sox11 prevents caspase-6 autocleavage.

<p>(A) Protein lysates were harvested and separated on a 4–12% bis-tris gel. Top panel shows a western blot of caspase-6 cleavage, the slower migrating band represents the uncleaved caspase-6 zymogen (note caspase-6 runs slighter higher than caspase-6 C163A because of an N-terminal flag tag) and the lower band represents caspase-6 following removal of its prodomain (lane 2). Cleavage of caspase-6 into caspase-6 Δprodomain is diminishedd in the presence of sox11 (lane 6). The lower panel is a western blot probed with an antibody capable of detecting the caspase-6 large subunit. Generation of the caspase-6 large subunit was suppressed in the presence of sox11 (compare lanes 2 and 4 with lanes 6 and 8, respectively). (B) Negative control experiments were performed using two other proteins identified in the yeast-two hybrid screen, COP1 and FEZ1. Neither FEZ1 nor COP1 affected caspase-6 self-cleavage. (C and D) Caspase-6 protein levels quantified using densitometry showed sox11 to significantly decrease caspase-6 protein levels by 30% but caspase-6 activity is completely blocked in the presence of sox11 (n = 8, unpaired t-test, **** p<0.0001).</p

Sox11 reduces caspase-3 and -7 activity but not caspase-8 or -9 activity.

<p>(A and C) Top panels show caspase-7 and caspase-3 expression levels with and without sox11 co-expressed. Sox11 co-expression with caspase-3 appears to affect its levels more than caspase-7. The second panel shows the large subunit fragments cleaved from caspase-7 and caspase-3. Both caspase-3 and 7 self- cleavage are reduced. The bottom two panels show sox11 was successfully co-expressed and actin levels indicate protein loading was equal. (B and D) Caspase-3/7 substrate luminescent based assay showed sox11 to reduce both caspase-3 and 7 activities to almost control levels. One-way anova, Sidak’s multiple comparsion test, ****p<0.0001 (E and G) Sox11 co-expression with either caspase-8 or 9 does not affect their steady state levels or proteolysis. (F and H) Caspase-8 and 9 activity assays showed no significant effect of sox11 (n = 3, one-way anova, Sidak’s multiple comparsion test).</p

Nuclear localization of sox11 contributes to the loss of caspase-6 activity.

<p>(A i-ix) Immunofluorescent images of caspase-6 and sox11, as well as caspase-6 and sox11 deletion mutants captured using a confocal microscope. Several sox11 mutants lose their nuclear localization i.e. (v), (vii), and (ix). Neither sox11 nor sox11 mutants altered the distribution of caspase-6 within the cell. (B) Microscopy images taken to compare the cellular distribution of sox11 and sox11 with the deletion of a putative NLS. Sox11 ΔNLS loses its nuclear localization and becomes more cytoplasmic (lower panel). (C) Western blot comparing caspase-6 proteolysis in the presence of sox11 versus sox11 ΔNLS. Sox11 ΔNLS is no longer capable of completely blocking caspase-6 cleavage (compare lanes 5 and 6). (D) Quantification of caspase-6 FL (full length, zymogen) levels vs caspase-6 post prodomain cleavage with and without sox11 ΔNLS from (C). No significant change was seen in caspase-6 full length levels in the presence of sox11 ΔNLS. However, caspase-6 prodomain levels were increased 56% in the presence of sox11 ΔNLS.</p

Confirmation of positive interactions in yeast and cells.

<p>(A) Bait (caspase-6 construct) and candidate prey (sox11 construct from library screen) were cotransformed into yeast and plated on selective media. Both bait and candidate prey plasmids can grow on double drop-out plates. Only colonies resulting from a genuine protein-protein interaction can grow on quadruple drop-out plates. Positive clones are circled in white. As expected, the negative control (vector+sox11) produced no colonies with quadruple drop-out. (B) Illustration of caspase-6 and the constructs used for co-immunoprecipitation. Yellow and orange bars denote propeptide. (C) Caspase-6 was immunoprecipitated from lysates using anti-GFP tagged caspase-6 subunits alone and with sox11. Lysates were immunoprecipitated using anti-GFP and probed with anti-flag. Top panel shows an interaction of the large caspase-6 subunit catalytically inactive with flag-tagged sox11 (Lane 7). The lower panel shows GFP tagged proteins were successfully imunoprecipitated. * indicates IgGs.</p

Regions within sox11 contributing to caspase-6 activity decreases include amino acids 117–214, as well as the c-terminal transactivation domain.

<p>(A) Schematic diagram depicting sox11 deletion mutants. (B) Caspase-6 substrate assay show all deletions within sox11 caused a significant reduction in caspase-6 activity. Although sox11 mutants Δ117–187, Δ188–214, Δ117–214, ΔC33, and ΔC33 Δ117–214 appeared to cause an increased trend in caspase-6 activity, no significant increase was found when compared to wildtype sox11 (n = 3, one-way anova, Sidak’s multiple comparsion test Sox11 mutants containing deletions of amino acids 6–49, 214–296, or deletion of either the HMG domain or polyserine region retained their capacity to retard caspase-6 activity(C) Western bolt analysis of sox11 mutants (upper panel) show a large difference in expression patterns. Western blots of caspase-6 proteolysis revealed that the sox11 mutants which did not reduce caspase-6 activity to control levels were also incapable of preventing caspase-6 cleavage (lanes 7, 8, 10, 12, 13). (D) Sox11 and mutants were immunoprecipitated from lysates using anti-flag antibody. Caspase-6 was detected using anti-caspase-6 raised against the N-terminus of caspase-6.</p