Genomic structure and expression of Jmjd6 and evolutionary analysis in the context of related JmjC domain containing proteins

<p>Abstract</p> <p>Background</p> <p>The <it>jumonji C (JmjC) domain containing gene 6 </it>(<it>Jmjd6</it>, previously known as phosphatidylserine receptor) has misleadingly been annotated to encode a transmembrane receptor for the engulfment of apoptotic cells. Given the importance of JmjC domain containing proteins in controlling a wide range of diverse biological functions, we undertook a comparative genomic analysis to gain further insights in <it>Jmjd6 </it>gene organisation, evolution, and protein function.</p> <p>Results</p> <p>We describe here a semiautomated computational pipeline to identify and annotate JmjC domain containing proteins. Using a sequence segment N-terminal of the Jmjd6 JmjC domain as query for a reciprocal BLAST search, we identified homologous sequences in 62 species across all major phyla. Retrieved <it>Jmjd6 </it>sequences were used to phylogenetically analyse corresponding loci and their genomic neighbourhood. This analysis let to the identification and characterisation of a bi-directional transcriptional unit compromising the <it>Jmjd6 </it>and <it>1110005A03Rik </it>genes and to the recognition of a new, before overseen <it>Jmjd6 </it>exon in mammals. Using expression studies, two novel <it>Jmjd6 </it>splice variants were identified and validated <it>in vivo</it>. Analysis of the <it>Jmjd6 </it>neighbouring gene <it>1110005A03Rik </it>revealed an incident deletion of this gene in two out of three earlier reported <it>Jmjd6 </it>knockout mice, which might affect previously described conflicting phenotypes. To determine potentially important residues for <it>Jmjd6 </it>function a structural model of the Jmjd6 protein was calculated based on sequence conservation. This approach identified a conserved double-stranded β^-helix (DSBH) fold and a HxDx_nH facial triad as structural motifs. Moreover, our systematic annotation in nine species identified 313 DSBH fold-containing proteins that split into 25 highly conserved subgroups.</p> <p>Conclusion</p> <p>We give further evidence that <it>Jmjd6 </it>most likely has a function as a nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenase as previously suggested. Further, we provide novel insights into the evolution of Jmjd6 and other related members of the superfamily of JmjC domain containing proteins. Finally, we discuss possibilities of the involvement of <it>Jmjd6 </it>and <it>1110005A03Rik </it>in an antagonistic biochemical pathway.</p

The bioluminescent Listeria monocytogenes strain Xen32 is defective in flagella expression and highly attenuated in orally infected BALB/cJ mice

Abstract Background In vivo bioluminescence imaging (BLI) is a powerful method for the analysis of host-pathogen interactions in small animal models. The commercially available bioluminescent Listeria monocytogenes strain Xen32 is commonly used to analyse immune functions in knockout mice and pathomechanisms of listeriosis. Findings To analyse and image listerial dissemination after oral infection we have generated a murinised Xen32 strain (Xen32-mur) which expresses a previously described mouse-adapted internalin A. This strain was used alongside the Xen32 wild type strain and the bioluminescent L. monocytogenes strains EGDe-lux and murinised EGDe-mur-lux to characterise bacterial dissemination in orally inoculated BALB/cJ mice. After four days of infection, Xen32 and Xen32-mur infected mice displayed consistently higher rates of bioluminescence compared to EGDe-lux and EGDe-mur-lux infected animals. However, surprisingly both Xen32 strains showed attenuated virulence in orally infected BALB/c mice that correlated with lower bacterial burden in internal organs at day 5 post infection, smaller losses in body weights and increased survival compared to EGDe-lux or EGDe-mur-lux inoculated animals. The Xen32 strain was made bioluminescent by integration of a lux-kan transposon cassette into the listerial flaA locus. We show here that this integration results in Xen32 in a flaA frameshift mutation which makes this strain flagella deficient. Conclusions The bioluminescent L. monocytogenes strain Xen32 is deficient in flagella expression and highly attenuated in orally infected BALB/c mice. As this listerial strain has been used in many BLI studies of murine listeriosis, it is important that the scientific community is aware of its reduced virulence in vivo

Genomic structure and expression of Jmjd6 and evolutionary analysis in the context of related JmjC domain containing proteins

<p>Abstract</p> <p>Background</p> <p>The <it>jumonji C (JmjC) domain containing gene 6 </it>(<it>Jmjd6</it>, previously known as phosphatidylserine receptor) has misleadingly been annotated to encode a transmembrane receptor for the engulfment of apoptotic cells. Given the importance of JmjC domain containing proteins in controlling a wide range of diverse biological functions, we undertook a comparative genomic analysis to gain further insights in <it>Jmjd6 </it>gene organisation, evolution, and protein function.</p> <p>Results</p> <p>We describe here a semiautomated computational pipeline to identify and annotate JmjC domain containing proteins. Using a sequence segment N-terminal of the Jmjd6 JmjC domain as query for a reciprocal BLAST search, we identified homologous sequences in 62 species across all major phyla. Retrieved <it>Jmjd6 </it>sequences were used to phylogenetically analyse corresponding loci and their genomic neighbourhood. This analysis let to the identification and characterisation of a bi-directional transcriptional unit compromising the <it>Jmjd6 </it>and <it>1110005A03Rik </it>genes and to the recognition of a new, before overseen <it>Jmjd6 </it>exon in mammals. Using expression studies, two novel <it>Jmjd6 </it>splice variants were identified and validated <it>in vivo</it>. Analysis of the <it>Jmjd6 </it>neighbouring gene <it>1110005A03Rik </it>revealed an incident deletion of this gene in two out of three earlier reported <it>Jmjd6 </it>knockout mice, which might affect previously described conflicting phenotypes. To determine potentially important residues for <it>Jmjd6 </it>function a structural model of the Jmjd6 protein was calculated based on sequence conservation. This approach identified a conserved double-stranded β^-helix (DSBH) fold and a HxDx_nH facial triad as structural motifs. Moreover, our systematic annotation in nine species identified 313 DSBH fold-containing proteins that split into 25 highly conserved subgroups.</p> <p>Conclusion</p> <p>We give further evidence that <it>Jmjd6 </it>most likely has a function as a nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenase as previously suggested. Further, we provide novel insights into the evolution of Jmjd6 and other related members of the superfamily of JmjC domain containing proteins. Finally, we discuss possibilities of the involvement of <it>Jmjd6 </it>and <it>1110005A03Rik </it>in an antagonistic biochemical pathway.</p

Extending the host range of Listeria monocytogenes by rational protein design

SummaryIn causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis

Influence of Internalin A murinisation on host resistance to orally acquired listeriosis in mice

Background: The bacterial surface protein internalin (InlA) is a major virulence factor of the food-born pathogen Listeria monocytogenes. It plays a critical role in the bacteria crossing the host intestinal barrier by a species-specific interaction with the cell adhesion molecule E-cadherin. In mice, the interaction of InlA with murine E-cadherin is impaired due to sequence-specific binding incompatibilities. We have previously used the approach of ‘murinisation’ to establish an oral listeriosis infection model in mice by exchanging two amino acid residues in InlA. This dramatically increases binding to mouse E-cadherin. In the present study, we have used bioluminescent murinised and non-murinised Listeria strains to examine the spatiotemporal dissemination of Listeria in four diverse mouse genetic backgrounds after oral inoculation. Results: The murinised Listeria monocytogenes strain showed enhanced invasiveness and induced more severe infections in all four investigated mouse inbred strains compared to the non-murinised Listeria strain. We identified C57BL/6J mice as being most resistant to orally acquired listeriosis whereas C3HeB/FeJ, A/J and BALB/cJ mice were found to be most susceptible to infection. This was reflected in faster kinetics of Listeria dissemination, higher bacterial loads in internal organs, and elevated serum levels of IL-6, IFN-γ, TNF-α and CCL2 in the susceptible strains as compared to the resistant C57BL/6J strain. Importantly, murinisation of InlA did not cause enhanced invasion of Listeria monocytogenes into the brain. Conclusion: Murinised Listeria are able to efficiently cross the intestinal barrier in mice from diverse genetic backgrounds. However, expression of murinized InlA does not enhance listerial brain invasion suggesting that crossing of the blood brain barrier and crossing of the intestinal epithelium are achieved by Listeria monocytogenes through different molecular mechanisms